We synthesize the gene of interest (GOI) into an expression vector with the selected features (expression host, affinity tag, signal sequence, etc.). Please download and fill out our questionnaire for custom-specific gene synthesis and cloning, it will help us learn more about your project and requirements.

Requirements for IBA’s custom services:
The customer is requested to provide the complete gene sequence of interest in electronically readable form or its “Genbank“ accession number. Any information concerning the biochemistry of the protein of interest would be helpful for planning a suitable expression strategy.

Alternative procedure – StarGate Cloning

The use of the StarGate cloning system is of advantage for expression projects, where the best expression features (affinity tags, promoters or expression host) for the gene of interest are unknown.

This service comprise the synthesis of the desired gene into StarGate^® Entry Vectors and subsequently the transfer into the StarGate Acceptor Vectors (providing a variety of expression features) with optimal control of expression in E. coli and mammalian cells.

We will deliver custom products specifically designed to support unique protein production needs from

• cloning desired DNA into an expression vector with selected features, to
• protein expression of the desired construct in E. coli and HEK293 cells, and
• protein purification via Twin-Strep-tag^®

Custom protein expression in E.coli

Production Phase 

Custom protein expression in mammalian cells

Test Phase 

Production Phase 

Strep-Tactin^®XT has a high binding affinity with Twin-Strep-tag^® in low picomolar range. Therefore, it is highly suitable for screening for therapeutic proteins and industrial enzymes as well assay development. For assay development we offer unconjugated Strep-Tactin^®XT.

For requests, please contact us

MHC I Streptamer^® custom production

You cannot find the combination of MHC I allele and antigen you need for your experiment? Or you need an entirely new MHC I allele and/or antigen that you did not see in our current portfolio? We produce customized MHC I-Streps with the alleles and antigens according to your needs. If you already have your own peptide that you would like to use, just mention it in our questionnaire, otherwise we can synthesize the peptide for you.

Please contact us for further information:

Protein Affinity Chromatography

The isolation of a target protein from a complex mixture is a precondition for analyzing the protein’s function without interference from other molecules, making protein purification one of the main applications in biotechnology. A target protein can be isolated from other molecules most efficiently by its affinity for another molecule, since affinity is a unique property. It is also possible to use other properties, such as molecular mass, charge, or hydrophobicity, although these properties can be largely similar between different proteins.

To purify a protein with the help of their affinity to another molecule, the interacting partner (ligand), e.g., a protein, a small molecule, or a metal, is immobilized on the stationary phase of the chromatography matrix. The stationary phase mostly consists of agarose or synthetic polymers and is packed into a column in the form of beads. The beads are surrounded and moisturized by a liquid, called the mobile phase. When the target protein-containing sample is applied, it enters the mobile phase and runs through the beads of the stationary phase. Meanwhile, the target protein can bind to the ligand, whereas other molecules remain in the mobile phase and can be removed by washing. For elution of the target protein, the interaction to the ligand is resolved by changing the buffer conditions, for example, the pH value, or by adding a specific competitor that supplants the target protein from the ligand resulting in dissociation of the target, while the ligand remains immobilized on the stationary phase.

Such a direct affinity purification strategy is most commonly used for antibodies based on antigen-antibody interactions. One example is protein A (ligand), which can be utilized to purify immunoglobulin G antibodies (target protein). But how can a protein be purified if an interaction partner is not known so far? In this case, the affinity of known interactions can be utilized for the indirect capture of the target protein. For this purpose, the target protein is tagged with a short peptide of one of the known interactors, which can bind to the other interaction partner immobilized on the stationary phase. The short peptide of the known interactor is called affinity tag and can be, for instance, the His-tag, GST-tag, Strep-tag®II, or Twin-Strep-tag®. They can bind to ligands such as metal ions, glutathione, Strep-Tactin®, or Strep-Tactin®XT, respectively.

Principle of affinity-based protein purification

Comparison of direct and indirect affinity purification strategies

Due to the highly specific selection and the resulting purity of target proteins, affinity-based systems have become the flagship method of purification. However, some of the widely used affinity tags, such as the His-tag, exhibit several drawbacks and limitations, since they can increase the risk of distorting the natural conformation of the target protein or necessitate stringent elution and wash conditions affecting the yield of the target protein. Additionally, many tags are not compatible with varying buffer conditions and must be removed to not impair downstream processing.

The widely used Strep-tag® technology, consisting of two engineered streptavidin variants, Strep-Tactin® and Strep-Tactin®XT, and two affinity tags, Strep-tag®II and Twin-Strep-tag®, is not limited in the use of buffers and its high specificity leads to obtaining highly pure proteins.

Therefore, IBA provides several resins, either coupled with Strep-Tactin® or Strep-Tactin®XT, which are all applicable for Strep-tag®II and Twin-Strep-tag® fusion protein purification. The purification cycle varies between both streptavidin variants, but both resin types serve the same goal: simple, fast, and variable protein purification procedures for highly pure proteins.

The following application notes provide comparison of Strep-Tactin® and Strep-Tactin®XT purification systems: for purification of Latex Clearing Protein , for further proteins (1, 2).

The special differences to other systems, especially between the His-tag system and Strep-tag® technology, are presented in a comprehensive comparison. It summarizes the differences as well as recommends one of the systems depending on the properties of the target protein, expression host, and purification conditions.

The differences between systems in protein purification from Expi supernatants is demonstrated in this application note.

“IBA’s unique Strep-tag® technology is a commonly used tool for the affinity purification of recombinant proteins and is based on one of the strongest non-covalent interactions in nature, which is the interaction of biotin to streptavidin. The system includes two affinity tags: Strep-tag®II and Twin-Strep-tag® (the tandem version of the Strep-tag®II). These peptide sequences exhibit intrinsic affinity towards two specifically engineered streptavidin variants — Strep-Tactin® and Strep-Tactin®XT.”

“The Strep-tag® technology stands for the highest protein purities under physiological conditions and can be used in a variety of different applications: in a field of purification, immobilization of proteins for assay development, and for protein interaction studies.”

Biotin blocking

Blocking of free biotin in cell culture supernatants and lysates

Cell culture supernatants often contain high amounts of free biotin. These are unproblematic when working with Strep-Tactin®XT resins, since biotin does neither bind irreversibly to this ligand nor reduces the binding capacity. However, this is the case for the application of resins coupled to Strep-Tactin®. To prevent the impairment of Strep-Tactin®, BioLock, which contains avidin, can be applied to mask free biotin. Avidin is a homolog of streptavidin and can be found in the egg white of birds, reptiles, and amphibians. It consists of four subunits, each of which can bind one molecule of biotin. In contrast to the streptavidin variants, Strep-Tactin® and Strep-Tactin®XT, it is not able to bind Strep-tag®II or Twin-Strep-tag® fusion proteins. Therefore, avidin can be used to selectively mask free biotin in cell lysates or cell culture supernatants, whereas the Strep-tag®II or Twin-Strep-tag® at the target protein remain accessible. An overview of media containing biotin and cell-internal biotin pools is given in the manual for biotin blocking. Blocking of biotin by BioLock is simple and fast. Only a small amount has to be added and, after a short incubation, the sample is ready for protein purification.

Blocking of biotinylated proteins

Besides free biotin, cell lysates also contain small amounts of biotinylated proteins. Normally, these proteins do not influence the purification results with Strep-Tactin® or Strep-Tactin®XT due to their low abundance. However, when it comes to analytic applications with high sensitivity, impurities should be prevented. One option is the treatment of the sample with BioLock prior to protein purification. Otherwise, if the target protein is already purified and should be detected via western blot with Strep-Tactin® or Strep-Tactin®XT conjugates, the application of Biotin Blocking Buffer is possible. Just as BioLock, Biotin Blocking Buffer contains avidin to mask biotin and biotinylated proteins, but the buffer is especially optimized for western blots. Shortly before detection, the membrane is incubated with the diluted buffer for a few minutes. However, if the detection occurs with the aid of StrepMAB-Classic or StrepMAB-Immo, masking of biotinylated proteins is not necessary. In contrast to Strep-Tactin® or Strep-Tactin®XT conjugates, these antibodies do not bind to biotin or biotinylated proteins and therefore do not lead to unspecific signals.

Binding patterns of Strep-tag®II, Twin-Strep-tag® and biotin

High-Throughput Purification

Before a specific target protein can be singled out and investigated in detail, a large number of proteins have to be evaluated in high-throughput screenings (HTS). To focus on their function without the influences of the other molecules present in the organism, these proteins have to be purified. Many high throughput purification applications require expensive equipment and specific technical knowledge. Alternatively, well-known and already established methods can be scaled up to be carried out in any size of lab.

Manual applications

For small-scale experiments with a limited number of samples, spin columns can be used. They are a convenient tool for small sample volumes that can still be handled manually. Thereby the number of proteins that can be purified at the same time in a fast and efficient way is flexible. A Spin Column Kit with all necessary components for protein purification, as well as Empty Spin Columns that can be combined with the customer’s resin of choice are available from IBA Lifesciences.

Semi-automated applications

Magnetic beads, such as MagStrep® Strep-Tactin®XT beads, can be used in a round-bottom 96-well plate, allowing the simultaneous purification of up to 96 samples. In this format, the application can still be carried out manually, but the use of pipetting robots is also possible. The magnetic core of the beads allows the use of a magnet to fix the beads to the wells when removing the supernatant, diminishing the loss of beads and eliminating the need for centrifugation.

Automated applications

For an automated approach that requires minimal manual intervention, IMCStips® containing Strep-Tactin®XT resin are available. The tip format allows high-throughput affinity purification with consistency and reproducibility making it especially attractive to industrial users. The approach is described in detail in the following application note. You can learn more about the automated purification of proteins using IMCStips® on the IMCS website .

Protein purification workflow with IMCStips® containing Strep-Tactin®XT resin

Analytical High-throughput Screening Applications

The identified and purified proteins from the initial screening can then be further characterized by using the Strep-tag® technology. Due to the outstanding high affinity of the Twin-Strep-tag® to Strep-Tactin®XT (pM Range), Strep-Tactin®XT is very well suited for biomolecular interaction analysis technologies as Surface Plasmon Resonance (SPR) or Biolayer interferometry (BLI).

Protein purification product formats

Protein purification via affinity chromatography is based on the binding affinities between different molecules. During purification, target protein tagged with a short peptide binds to the ligand, which is usually coupled to resin, while unwanted proteins without tag do not bind and can be washed away.

The resin can consist of agarose or magnetic beads. Both come in suspension form. Alternatively, agarose beads can also be prepacked in FPLC, gravity or spin columns. Which format works best for your application depends either on the yield you require or on the initial sample volume.

Choosing the product format based on…

Yield

Sample volume

These two schemes should help you decide how to choose the right product format for protein purification among the four most popular formats: columns for FPLC/HPLC devices, gravity flow columns, magnetic beads and spin columns. Please note that the recommendation depends on whether you have the required yield or sample volume as a starting point.

Product formats in details

FPLC/HPLC columns

Large sample volumes can be purified using columns in automated FPLC/HPLC devices such as Äkta®. FPLC columns come with different resin volumes and can be connected in series to increase the binding capacity. They attach to the device via the standard 10-32 connectors.

• Broad pH compatibility
• Excellent pressure stability
• Outstanding target purity
• 1 & 5 ml column bed volume available

Product advantages:

• Quick application of large sample volumes
• Fully automated protein purification
• Time saving: No waiting between the purification steps, since the device applies the correct volumes of buffer and sample autonomously.
• Recording of the complete affinity chromatography protocol, which provides information about the purification quality
• Reproducible purification results due to the program settings that are set up beforehand and not dependent on the user

Gravity flow columns

Gravity flow columns are ideal for protein purifications with a medium yield. The resin suspensions allow you to pack your own columns according to your need, but you can also choose from other formats, like pre-packed columns.

• Optimized for column affinity chromatography
• Variety of different product sizes and matrices

Prepacked columns

Suspension

Magnetic beads

Magnetic beads can be used for batch purifications in tubes or 96-well plates to purify proteins in pull-downs, IPs and Co-IPs. The beads can be easily fixed to the reaction tube with a magnet, so you can skip centrifugation without having to worry about losing your sample during pipetting steps.

No centrifugation
Separation via magnetic separator
2 ml and 10 ml volume available

Spin columns

If you need to process multiple small samples in parallel, you can use spin columns with the resin of your choice. They allow you to do a batch purification of a different protein in every tube with a simple setup.

• Compatible with conventional 1.5 ml or 2.0 ml reaction tubes
• Buy a ready-to-use kit or fill empty columns with resin of your choice

Resin reuse

Strep-Tactin® and Strep-Tactin®XT resins for protein purification can be regenerated and reused >10 times* without loss in performance. Resin activation can easily be checked with HABA. The yellow HABA solution turns red (Strep-Tactin®) or orange (Strep-Tactin®XT) upon binding to the engineered biotin binding pockets of Strep-Tactin® and Strep-Tactin®XT clearly indicating that the resin is fully regenerated. Afterwards, HABA can be removed by washing with 100 mM NaOH. Once the red color has disappeared and NaOH was removed from the column using Buffer W, the column can be reused. If the biotin binding pocket is blocked or damaged no color shift occurs and the resin cannot be reused.

*under ideal conditions >50 regeneration cyles are possible

Resin specifications

Application example

Cell culture supernatant of MEXi-293E cells (24 ml) was spiked with 240 µg of a large protein (130 kDa) C-terminally tagged with the Twin-Strep-tag®. Protein purification was carried out with a 0.2 ml Strep-Tactin®XT 4Flow® high capacity gravity flow column. After sample application the column was washed with 1x Buffer W. The Twin-Strep-tag® protein was eluted by three elution steps with 0.7, 1.5 and 0.8 CV 1x Buffer BXT. Purification results were analyzed by SDS-PAGE (A) and western blot (B). Both show a molecular weight marker (M*, Precision Plus Protein™ Unstained Protein Standards, Strep-tagged recombinant (BioRad), M** PageRuler™ Plus Prestained Protein Ladder (Thermo Fisher Scientific)), the spiked cell supernatant (S), flow through (FT), the 1st and 5th wash fraction (W1 and W5, respectively) and the elution fractions (E1-E3). Strep-Tactin® HRP was applied (1:4000) for detection via western blot.

Protein purification with magnetic beads

Magnetic bead-based protein purification is a highly efficient method, particularly for small sample volumes or viscous materials. MagStrep® Strep-Tactin®XT beads are ideal for batch purification, offering easy separation from the supernatant and high specificity, resulting in exceptionally pure proteins. This method is versatile and scalable, suitable for different sample sizes and easily adaptable for automated systems, making it perfect for high-throughput screenings. Coated with the high-affinity ligand, Strep-Tactin®XT, these beads enable specific binding to strep-tagged proteins, providing a simple and customizable workflow for your experimental needs.

It is easy to increase protein yield by adjusting specific parameters when purifying strep-tagged proteins with magnetic beads. Discover these optimization steps below, including protein size, incubation time, bead volume, and protein concentration.

Pull-down assays with magnetic beads

Understanding protein-protein interactions is crucial for studying protein structure and function. Pull-down assays are essential techniques for detecting these interactions, either confirming predicted interactions or discovering new ones. The pull-down method is cost-effective and convenient, using a bait protein fused with an affinity tag, such as the Twin-Strep-tag®, and immobilized on magnetic beads coupled to the corresponding ligand, Strep-Tactin®XT.

Find out more about MagStrep® Strep-Tactin®XT beads, that offer low pM binding affinity with Twin-Strep-tagged bait proteins, ensuring efficient single-step purification of complexes.

From early screening to large scale protein production

Magnetic bead-based purification is highly scalable, making it suitable for processing a wide range of sample volumes. The process can be adapted to different sample sizes, from small-scale high-throughput screenings to large-scale protein production, depending on the application.

Learn more about the flexible scaling of protein purification with magnetic beads in this application note: From Early Screening to Large-Scale Production.

Optimization of magnetic bead-based protein purification

MagStrep® Strep-Tactin®XT beads are the ideal tool for quick and simple purification of strep-tagged proteins. They can be used for any type of protein, in small-scale experiments or in high-throughput purifications. Their magnetic core enables separation from the supernatant without centrifugation or the use of a column, making the batch purification workflow very straightforward. Additionally, MagStrep® beads can be used to study protein-protein interactions via pull-down assays. The fast and easy purification conserves even weak protein-protein interactions.

Specifications of the MagStrep® Strep-Tactin®XT beads

In the protocol and the following information on optimization of magnetic bead purification, “bead volume” refers to the total volume of beads. 20 µl of the 5% suspension contain 1 µl of beads.

Optimization parameters

IBA’s protocol for MagStrep® Strep-Tactin®XT beads describe the basic steps for magnetic bead protein purification. Depending on your experimental conditions, it’s possible to optimize the purification protocol and increase protein yield by adjusting specific parameters. The following tips and data below will help you optimize magnetic bead protein purification by adjusting bead volume, incubation time, and protein concentration.

1. Required bead volume depends on the protein size

When working with small proteins, more molecules will bind per bead compared to large proteins. Due to their bigger surface area, large proteins require more space to bind. As a result, the binding capacity may noticeably decline for proteins >90 kDa, which can lead to a lower yield.

To ensure a high yield for large proteins, increase the bead volume to provide a sufficient binding surface. The optimal bead volume for your specific protein can be determined by titration.

Figure 1: Protein yield decreases for large proteins. 250 µl samples of Twin-Strep-tag® fusion proteins of different sizes with a starting protein concentration of 1.7 nmol/µl were incubated with 5 µl beads. Protein purification was carried out according to IBA’s standard protocol. After elution, the total protein content was compared to the protein content at the beginning.

Figure 2: Protein yield of large proteins increases when higher bead volumes are used. 250 µl samples of a 140 kDa protein at 1.7 nmol/µl were incubated with different bead volumes for 10 minutes. Afterwards, the purification was performed according to IBA’s standard protocol. Protein content of the elution was compared to the starting protein content.

2. Increased bead volumes reduce the incubation time

To achieve a higher protein yield in a shorter incubation time, add an excess of beads in relation to the amount of protein and binding capacity of the beads (0.85 nmol/µl beads or 42.5 µg/µl of a 50 kDa protein). Best yield in relation to the bead volume used is reached if 5x more beads are added in relation to the maximum binding capacity. For example, if the total amount of protein is 85 μg, which theoretically could be bound by 2 μl of beads, add 5x 2 μl = 10 μl beads and incubate for 10 minutes to achieve the best yield.

The maximum yield may also be reached when using a lower bead volume, but the incubation time may be noticeably longer.

Figure 3: When a higher bead volume is used, protein yield can be increased at a short incubation time. 250 µl samples of a 30 kDa Twin-Strep-tag® fusion protein were incubated with different bead volumes for the same incubation time. Protein content of the elution was compared to the protein content at the start.

Figure 4: At a low bead volume, protein yield can be increased by incubating for a longer time period. 250 µl samples of a 30 kDa Twin-Strep-tag® fusion protein were incubated with the same bead volume for different time periods. Protein content of the elution was compared to the protein content at the start.

3. Sample protein concentration and bead volume must be balanced

An important factor during the purification is the ratio between bead volume and the concentration of target protein in the sample. Our experiments showed that higher target protein concentrations are preferable. Samples with low protein concentrations, like cell culture supernatants or low abundant proteins, should be concentrated before applying them to the magnetic beads to increase the yield. We recommend a protein concentration of at least 1 pmol/µl (or 50 ng/µl of a 50 kDa protein). If possible, measure the target protein concentration in your sample before purification and adjust the bead volume accordingly.
However, if concentrating your sample is not an option, using a high volume of beads is not beneficial. For low concentrated proteins, a reduction of the bead volume increases the yield. Bead volume and protein concentration must always be balanced.

Figure 5: Higher protein concentrations lead to higher protein yield. 250 µl of different concentrated samples of a 30 kDa Twin-Strep-tag® fusion protein were incubated with 5 µl beads for 10 minutes. IBA’s standard protocol was carried out afterwards and the final protein content of the elution was compared to the protein content at the start.

Figure 6: A high bead volume does not lead to a high yield when working with low protein concentrations. 250 µl sample containing 0.5 or 1 pmol/µl of a 30 kDa Twin-Strep-tag® fusion protein, were incubated with 1.6 or 5 µl beads for 10 minutes. IBA’s standard protocol was carried out afterwards, and the final protein content of the elution was compared to the protein content at the start.

FAQ about magnetic bead purification

Application example

GFP C-terminally tagged with Strep-tag®II (28 kDa) was expressed in E. coli (A) and purified with MagStrep® Strep-Tactin®XT beads (MagStrep “type3” XT beads). For separation of magnetic beads from residual solution, the Magnetic Separator was used. Before elution, the sample was split and the target protein was eluted either by application of 1x Buffer BXT containing biotin or by boiling (C, 5 min at 95 °C). Due to boiling, the Beat´s agarose melts and Strep-Tactin®XT is released, leading to a further peak at 14 kDa. Protein purification results were analyzed with the Agilent Bioanalyzer 2100 system. The example shows the specific binding properties of MagStrep® Strep-Tactin®XT beads (MagStrep “type3” XT beads) and the high purity that can thereby be observed.

Reliable pull-down assays with MagStrep® Strep-Tactin®XT beads

Knowing which proteins interact with one another is an important step in understanding signaling pathways and specific protein functions, which is key for unraveling mechanisms behind diseases and identifying new targets for drug development. For studying protein-protein interactions, pull-down assays have become an invaluable tool for scientists. In pull-down assays, interactions between two or more proteins are detected, either confirming the predicted protein-protein interaction or discovering new ones. Pull-down approaches represent convenient and low cost in vitro techniques, in which the bait protein is fused to an affinity tag, such as the Twin-Strep-tag®, and immobilized on beads coupled to the corresponding affinity ligand, e.g. Strep-Tactin®XT resin. Afterwards, a protein source containing putative prey is applied. Alternatively, cell lysates containing the tagged bait protein and its potential interaction partners are directly added to the affinity resin. In both cases, the formed bait-prey complexes are eluted with excess chemical or biological reagents releasing the tag from the ligand.

In pull-down assays, interaction partners are “pulled down” along with the tagged protein during purification. Existing protein interactions are preserved during gentle washing steps. In the resulting elution, both the target and its interaction partners are present. Which proteins interacted can be identified through further analysis, for example in mass spectrometry.

MagStrep® Strep-Tactin®XT beads bind to bait proteins tagged with Twin-Strep-tag® with a picomolar affinity, while still maintaining binding reversibility and mild recovery. The specific binding of the Twin-Strep-tag® to Strep-Tactin®XT provides the basis for efficient single-step purification of tagged bait-prey complexes in vitro. Further, the Twin-Strep-tag® is small and chemically inert and thus does neither interfere with proper protein folding and function nor binding interactions in complexes. Mild and adjustable wash conditions ensure that the protein complexes remain intact. Another benefit is the mild elution, as no pH shift or harmful compounds are needed. By simply adding biotin, bait-prey complexes are specifically eluted, reducing co-purification of false positives to almost zero. These characteristics distinguish the Twin-Strep-tag® from other protein tags that are commonly used for pull-down assays such as FLAG-tag, HA-tag or Myc-tag. These epitope tags guarantee efficient immobilization due to an antibody-mediated binding, but the antibodies may also bind non-specific proteins with sequence similarity, leading to false-positive results. In order to achieve a specific elution of epitope tagged proteins, peptides are used in high concentrations, which is very expensive. Alternatively, pH-shift elution is possible, but also elutes unspecific bound proteins. By combining high affinity and high specificity with mild elution conditions, the Twin-Strep-tag® provides a perfect solution for efficient and reliable pull-down assays.

How to circumvent common problems associated with the His-tag system?

The His-tag (6xHis-tag, His6-tag or polyhistine-tag) is a popular affinity tag for protein purification since its small size ensures a low impact on protein structure. While the His-tag system is popular for its high yield and low costs, the time and effort it takes to establish successful His-tag purification is often neglected.

The following problems are commonly associated with the His-tag system:

• Low target purity due to unspecific binding of host proteins (Figure 1A)
• Sample or buffer components interfere with protein binding
• Protein precipitation after purification due to incompatible buffer components
• Culture media for mammalian cells interfere with protein binding or cause Ni2+ leakage
• Required pH for target protein causes elution from Ni-NTA agarose
• Limited applicability in protein analysis due to low tag-ligand affinity and specificity

To avoid or address these problems, careful planning of the procedure and optimization of the His-tag purification protocol is required. In the end, a His-tag might still not achieve satisfactory results or might not be suitable at all for a certain type of protein. In this case, a different affinity tag should be considered.

The Strep-tag® system is the ideal alternative to His-tag since all common problems are avoided. Due to the specific tag-ligand interaction, protein isolation using this system leads to highly pure protein without further optimization or processing steps (Figure 1B).

Figure 1: GFP fused to either a 6xHis-tag (A) or to a Twin-Strep-tag® (B) was isolated from E. coli lysates using standard protocols provided by the manufacturers for the used resins (Ni-NTA from Thermo Fisher Scientific for His-tag and Strep-Tactin®XT 4Flow® high capacity from IBA Lifesciences for Twin-Strep-tag®)

Watch our free on-demand about common problems during His-tag purification and read our white paper comparing His-tag and Strep-tag® protein purification systems.

What are causes of impurities during His-tag purification and how to eliminate contaminants?

His-tag is bound by transition metal ions, of which nickel (Ni2+) is most commonly used. Nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) usually serve as carrier matrices for Ni2+. Within the His-tag purification system, which is a type of immobilized metal affinity chromatography (IMAC), both the type of tag and the corresponding tag-binding metal ions (e.g. Ni2+, cobalt (Co2+) or copper (Cu2+)), potentially cause impurities:

Figure 2: Since His-containing proteins non-specifically bind to Ni-NTA resins, titration of wash and elution buffers are necessary. In addition, further processing steps such as e.g. size exclusion chromatography or filtration can be required to increase the purity sufficiently for further experiments. During this step, protein is lost, reducing the overall protein yield. In contrast, Strep-tag®II and Twin-Strep-tag® bind to their ligands with a high specificity, resulting in a highly pure protein preparation that can directly be used for further downstream applications.

• “His” or “Histidine” is an amino acid that may be present in host proteins. Consecutive His-residues mediate unspecific binding to the chelated metals on the His-tag purification resin.
• Host proteins may have an intrinsic affinity to Ni2+ or Co2+.

To increase the purity of His-tag proteins, several steps before, during and after the purification process offer optimization potential.

As a first step, elution agent imidazole can be added in a low concentration to the cell lysis buffer and to wash buffers. This way, unspecific binding of intrinsic proteins with His-tag can be prevented. However, the imidazole concentration that inhibits only the binding of His-containing host proteins and still allows the binding of 6xHis-tagged target proteins has to be carefully titrated. Similarly, gradually adjusting the imidazole concentration during elution can help to eliminate His-tag contaminants. If these approaches are not successful, increasing the number of His-residues from e.g. 6 to 10 achieves stronger binding of the target protein and permits higher imidazole concentrations during washing to remove contaminants. However, this means starting from scratch again, since protein expression has to be adapted.

If the purity of the His-tag target protein is not satisfactory after modifying the purification procedure, additional processing steps can be considered. These include size exclusion or ion exchange chromatography, filtration or dialysis. The disadvantages here are that all extra steps can cause protein loss and additional costs (Figure 2).

In contrast, the two tags that are used in Strep-tag®-based protein purification (Strep-tag®II and Twin-Strep-tag®) were specifically engineered for use in this technology. Therefore, unlike His residues, it is not likely that they naturally occur in proteins and subsequently bind to the purification resin (Figure 3). If any impurities occur during Strep-tag® purification, this is usually caused by biotinylated proteins, which are easily and specifically blocked with avidin. Otherwise, ready-to-use wash and elution can be applied as recommended.

Figure 3: E. coli lysate was added to Ni-NTA (A) or to Strep-Tactin®XT 4Flow® high capacity (HC) resin (B). Contaminants were only observed with Ni-NTA resin due to unspecific binding of proteins containing His residue.

Which reagents are compatible with the His-tag system?

The choice for or against a specific protein tag is strongly influenced by the properties of the target protein. Since pH values ranging from 4.5 to 6 and chelating agents such as EDTA lead to elution of His-tagged proteins, this system is not suitable for e.g. pH-sensitive proteins. The use of chelating agents also means that the transition metal ions are detached from the matrix and have to be recharged after purification. Furthermore, reagents such as DTT (reducing agent), Tris or MOPS are not recommended for the His-tag system, which further limits the broad applicability for different proteins. In addition to unwanted elution of proteins, the use of an incompatible reagent can lead to the formation of protein precipitates. In contrast, Strep-Tactin®XT resins are compatible with various reagents, making it a very flexible system for various protein classes (see table 1 for a detailed overview of compatible reagents). This way, it is suitable for e.g. membrane proteins, antibodies and metal ion containing proteins (e.g. metalloproteases).

Table 1: Compatible reagents and representative values for His- or Strep-tag® based protein purification.

Which applications are possible with the His-tag?

A prerequisite for flexible application options is the specificity and affinity of a tag-ligand interaction. The His-tag system disposes only an affinity in the µM-nM range. This affinity leads to rapid dissociation and poor immobilization. In addition, His-tag antibodies have only a low specificity and can also detect unspecific proteins with His residues arranged in tandem. A large number of analytical applications for which a high affinity and/or highly specific antibodies are necessary – such as SPR (Surface Plasmon Resonance), BLI (Bio-Layer Interferometry) – can only be addressed inadequately. Especially for kinetic measurements of high affinity interactions, the nanomolar affinity of His6-tag to NTA is not always sufficient. To circumvent this problem, a second affinity tag can be fused to the target protein. On the one hand, this potentially affects the functionality of the protein and on the other hand, additional costs are generated. The Strep-tag® technology by contrast offers an affinity in the µM–pM range. Depending on the application, the appropriate affinity can be selected. Further on, a large number of products are already available for the Strep-tag® technology, which allows a direct transition from protein purification to analytical application.

Which expression host is compatible with the His-tag system?

E. coli is most frequently used as expression host for His-tagged proteins. The amount of contaminants that occur during His-tag purification from E. coli cultures greatly depend on factors such as media composition and other culture conditions, genetic background of the E. coli strain and the recombinant protein that is expressed. Consequently, protocol optimization for reducing impurities have to be performed for each recombinant protein separately.

Since E. coli, is not always suitable for protein expression, especially for more complex proteins that require posttranslational modifications, alternative expression systems are needed. However, yeast and insect cell media usually have an acidic pH, which interferes with the binding of His-tagged proteins to the IMAC resin. Moreover, media for yeast or mammalian cell cultivation often contain amino acids, such as histidine, glutamine or arginine, which compete with the His-tag for binding sites.

The following whitepaper shows the advantages of the Strep-tag® technology in comparison to the His-tag system within two high density mammalian expression systems, Expi293TM and ExpiCHOTM.

Another problem of isolating polyhistidine-tagged proteins from certain types of cell culture media is the stripping of nickel ions from the resin. For example, purifying His-tagged proteins from Expi supernatants with Ni-NTA resins is problematic and has been associated with a loss of target protein in the purification process.

This application note demonstrates that a standard Ni-NTA resin does not provide reliable and predictable purification results when used for protein purification from Expi supernatants. Furthermore, it shows that standard Ni-NTA resins exhibit significant reduction of binding capacity and recovery due to nickel leakage by media supplements.

Specifications of Strep-tag® Protein Purification Resins

There are several resin versions available for the purification of strep-tagged target proteins. All resins differ in their properties and suitability for applications. Learn more about the different resin matrices in order to know when to use which resin.

Resin reuse

Strep-Tactin® and Strep-Tactin®XT resins for protein purification can be regenerated and reused >10 times* without loss in performance. Resin activation can easily be checked with HABA. The yellow HABA solution turns red (Strep-Tactin®) or orange (Strep-Tactin®XT) upon binding to the engineered biotin binding pockets of Strep-Tactin® and Strep-Tactin®XT clearly indicating that the resin is fully regenerated. Afterwards, HABA can be removed by washing with 100 mM NaOH. Once the red color has disappeared and an addiotinal wash step using Buffer W was performed to remove NaOH, the column can be reused. If the biotin binding pocket is blocked or damaged no color shift occurs and the resin cannot be reused.

*under ideal conditions >50 regeneration cycles are possible

Find out more about Strep-tag® protein purification

Here, we have grouped links to make it easier to find more information about Strep-tag® technology for protein purification. In our web shop, you can find the protein purification products that fit your research needs. Read the comprehensive comparison of the most used tag systems, Strep-tag® vs. His-tag. Browser through application examples and frequently asked questions about Strep-tag® purification system or download application notes in our download area.

Cost-saving protein research due to simple & efficient regeneration of Strep-Tactin® and Strep-Tactin®XT

The ability to reuse purification or immobilization products saves both time and costs by reducing the need for frequent replacements. This benefit extends to a range of applications, including affinity chromatography, magnetic batch purification, and SPR/BLI. To allow for an effective re-use without loss of binding capacity, a regeneration procedure that is efficient but not detrimental to the binding ligand and its supporting material is required. Due to their binding properties as well as their high stability, Strep-Tactin® and Strep-Tactin®XT coupled to magnetic or non-magnetic agarose beads or to biosensor chips are perfectly suitable for multiple regeneration cycles and even clean-in-place (CIP) procedures.

Regeneration of affinity chromatography resins

Strep-Tactin® and Strep-Tactin®XT affinity chromatography resins are easily regenerated with 100 mM sodium hydroxide (NaOH). It is a simple procedure in which the regeneration buffer is directly applied after the final protein elution step. After washing the column, it can immediately be used for further purification steps or stored for later usage (Fig. 1). Since the ligands (Strep-Tactin® & Strep-Tactin®XT) are stably coupled to the agarose beads, resins can be regenerated 50 times* without losing its binding capacity (Fig. 2). For Strep-Tactin®XT resins, IBA Lifesciences also offers a ready-to-use regeneration buffer (Buffer XT-R) containing 3 M MgCl2 that can be used as effectively as 100 mM NaOH (Fig. 2 right).

*may vary depending on protein properties and required buffer components

Figure 1: Quick & easy regeneration principle for Strep-Tactin® & Strep-Tactin®XT resins. After eluting the bound protein, 100 mM NaOH can be applied for regeneration. After washing with Buffer W, the resin is ready for another purification cycle or for storage.

Figure 2: (left) Strep-Tactin® and (right) Strep-Tactin®XT affinity chromatography resins can be regenerated 50 times with 100 mM NaOH without loss in binding capacity. For Strep-Tactin®XT, a ready-to-use regeneration buffer (Buffer XT-R) containing 3 M MgCl2 is offered for convenient use.

Clean-in-place (CIP)

In bioprocessing applications, it is not only relevant that an affinity chromatography resin can be re-used multiple times to reduce costs, but also that it can be cleaned efficiently to prevent column fouling. Impurities need to be removed to avoid sample contamination as well as a decline in column performance. A standard cleaning agent in bioproduction is NaOH. A sufficiently high concentration of NaOH removes i.e. proteins, lipids, nucleic acids as well as bacteria. While highly stable at 100 mM NaOH (Fig. 2) Strep-Tactin® and Strep-Tactin®XT resins can also be exposed to 500 mM NaOH at least 10 times without affecting the resin performance (Fig. 3). Even after 50 cycles, only a marginal decline in binding capacity is observed, demonstrating the suitability of Strep-Tactin® and Strep-Tactin®XT resin for CIP procedures.

Figure 3: Strep-Tactin® as well as Strep-Tactin®XT resins can be exposed to 500 mM NaOH during several CIP cycles without a significant loss in binding capacity.

Regeneration of magnetic beads

Figure 4: MagStrep® Strep-Tactin®XT beads were regenerated with 100 mM NaOH after protein purification. The binding capacity remained stable over at least 10 regeneration cycles.

An alternative for affinity chromatography-based protein purification is batch purification using magnetic beads. Magnetic beads are especially popular for small-scale and high throughput applications. However, upscaling is also possible, which makes the option for regeneration key for a cost-effective usage. MagStrep® Strep-Tactin®XT beads are agarose-coated magnetic beads that can be re-used at least 10 times if regenerated with 100 mM NaOH after every purification (Fig. 4). Similar as for affinity chromatography resins, the regeneration procedure is simple and straightforward, requiring only two basic steps: incubation with NaOH and subsequent wash.

Figure 5: (left) Strep-Tactin®XT coated on biosensors enables a simple & straightforward regeneration procedure in analytical applications such as SPR and BLI. (right) A Strep-Tactin®XT sensor chip was regenerated with 3 M GuHCl after each measurement. The capture level of a Twin-Strep-tagged protein ligand remained stable over at least 30 regeneration cycles.

Regeneration of SPR chips

The properties of Strep-Tactin®XT not only enable efficient regeneration in protein purification approaches, but also in analytical applications such as SPR or BLI. For kinetic measurements, Strep-Tactin®XT is immobilized on biosensors. After capturing a strep-tagged ligand and measuring the binding kinetics of an analyte, addition of 3 M guanidine hydrochloric acid (GuHCl) efficiently removes all ligand-analyte complexes. After GuHCl is washed away with an appropriate running buffer, another sample can be analyzed or the biosensor stored until further use (Fig. 5 left). With this simple procedure, Strep-Tactin®XT coated chips can be re-used at least 30 times without any negative effects on the capture level of the ligand (Fig. 5 right). In addition to saving costs, this option of easy regeneration is highly convenient for measuring several samples consecutively, since Strep-Tactin®XT remains stably bound to the biosensor after every cycle.

Surface Plasmon Resonance (SPR)

Surface plasmon resonance (SPR) is an optical biosensor application for direct, real-time, and label-free measurements. It allows the determination of protein concentrations as well as the analysis of affinity and kinetics of a specific protein to other proteins, DNA, RNA, or cells. Therefore, SPR is an innovative tool for biomolecular interaction studies and plays an essential role in biotherapeutic drug discovery and development. The principle of SPR is an optical measurement that detects binding of a biomolecule in solution (analyte) to a biomolecule that is immobilized on a gold-layered surface of a biosensor chip (ligand). Thereby, changes in the refractive index are determined which are proportional to the concentration of bound analyte at the surface of the biosensor chip.

A crucial step in SPR analysis is the immobilization of the ligand to the surface of the biosensor chip. The immobilization of the ligand can take place via direct covalent coupling to the surface of the chip or by capture with the help of a pre-coated capture molecule. However, the direct covalent immobilization of the ligand can change its biological activity or result in an undirected immobilization with reduced accessibility of the binding sites. The transient immobilization of tagged ligands via affinity capturing molecules overcomes these drawbacks.

Surface plasmon resonance (SPR) with Strep-Tactin®XT coated sensor chips

Capturing Twin-Strep-tag® proteins with BiacoreTM

Nevertheless, tags used for such applications must bind efficiently to the capturing molecule with an affinity that is higher than that between analyte and ligand. His-tagged ligands that are immobilized on an NTA-surface with a KD in the nanomolar range can be used for measuring weaker interactions, but they fail in the measurement of high-affinity interactions with slow dissociation rates. In contrast, Strep-Tactin®XT forms an exceptionally strong complex with the Twin-Strep-tag®. An affinity in the low picomolar range allows measurements of long dissociation times and slow off-rates.

Thus, the combination of these two components of the Strep-tag® technology enables a directed, high-affinity immobilization that does not influence the activity of the ligand or require any modifications. Due to the high-affinity interaction and its high specificity, non-specific binding of host cell proteins is avoided, and ligands can be captured efficiently and directly from culture media. Moreover, Strep-Tactin®XT is compatible with many substances and the biosensors can be easily regenerated. The Twin-Strep-tag® Capture Kit provides all necessary products for the preparation of Strep-Tactin®XT-coated biosensor chips and the following measurements.

Twin-Strep-tag® – a reliable and time saving alternative to Avi-tag in Surface Plasmon Resonance (SPR) analysis

Surface plasmon resonance (SPR) is a powerful method to study biomolecular interactions. Due to its high accuracy and ability to measure samples in high throughput, it plays a major role in research and development of drugs.

A common tag which is frequently used to immobilize ligands on SPR biosensor chips is the Avi-tag. It is characterized by its high binding affinity and specificity for streptavidin. The combination of Avi-tag and streptavidin allows long-term measurements of interactions with high affinity and slow dissociation rates.

Successful SPR measurements require a sufficient amount of highly pure ligand. Although its binding to streptavidin is very strong, the Avi-tag is not an ideal tag for initial purification of a chosen ligand. The available resins for purifying Avi-tag fusion proteins are expensive and only yield a low amount of protein. The solution for this is the use of a second, more suitable, tag for initial ligand purification. Due to its high purity in protein purification, as well as higher yield and lower costs, the Twin-Strep-tag® is often considered as second tag. Therefore, a frequently asked question is, if the Twin-Strep-tag® and the Avi-tag can be used on the same protein.

The answer is: Twin-Strep-tag® and Avi-tag should not be used on the same protein! The biotinylation of Avi-tag can interfere with the binding of Twin-Strep-tag®, since the binding partner of Twin-Strep-tag® is a streptavidin mutant called Strep-Tactin®XT. Strep-Tactin®XT still has an affinity to biotin and biotinylated proteins, which can affect SPR measurements. Conversely, Twin-Strep-tag® still has a weak affinity to streptavidin, which might influence SPR measurements as well.

The solution to the incompatibility of Avi-tag and Twin-Strep-tag® is quite simple. Avi-tag is not needed for SPR measurements, since Twin-Strep-tag® can be used just as effectively for protein immobilization. In addition to the suitability for the whole procedure from purification to immobilization, using Twin-Strep-tag® also has additional benefits:

• Strep-Tactin®XT has a pM affinity for Twin-Strep-tag® and therefore, the combination is ideally suited for analysis of interactions with high affinity and slow dissociation rates.
• The reversibility of the interaction allows reuse of the sensor chips
• Efficient and easy one-step purification method – no tedious additional biotinylation

As a result, using the Twin-Strep-tag®/Strep-Tactin®XT system is cost-efficient and time-saving and the preferred choice when planning kinetic measurements such as SPR.

Fig. 1: Affinity-based ligand capture with biotinylated Avi-tag/Streptavidin or Twin-Strep-tag®/Strep-Tactin®XT

Twin-Strep-tag^® – direct and time-saving protein purification with high yields

Fig. 2: Protein analysis workflow with Twin-Strep-tag® and Avi-tag

The ease of use, the high purity and yield are all features that indicate that Twin-Strep-tag® is superior to Avi-tag for protein purification. On top of that, the high affinity to Strep-Tactin®XT offers the possibility for efficient ligand immobilization necessary for SPR or other analyses of binding kinetics, making the Avi-tag essentially redundant.

Twin-Strep-tag® provides pM affinity to study high affinity interactions

SPR analysis requires immobilization of a ligand on a biosensor chip. To achieve this, the Avi-tag/streptavidin interaction is commonly used, because the binding is characterized by an exceptionally high affinity. Another interaction that offers the possibility for efficient immobilization is the binding of Twin-Strep-tag® to Strep-Tactin®XT. With an affinity in a low picomolar range it can achieve similar results in SPR analyses as the Avi-tag/streptavidin combination. The half-life of Twin-Strep-tag®/Strep-Tactin®XT complex is 13 hours, enabling measurements of high affinity interactions over a long period of time.

The versatility of the Twin-Strep-tag®/Strep-Tactin®XT combination during purification and analyses makes it a great alternative to using the Avi-tag/streptavidin interaction. By choosing the Twin-Strep-tag®, the whole workflow will become a more time-saving and cost-effective process.

Comparable results with Twin-Strep-tag® and Avi-tag for analysis of high affinity interactions

Fig. 3: In a direct comparison, CD45 was fused to either a Twin-Strep-tag® (TST) or an Avi-tag (Avi) and immobilized using a CM5 sensor chip coated with Strep-Tactin®XT or a Sensor Chip CAP coated with streptavidin. 2.56, 6.4, 16, 40, and 100 nM anti-CD45 antibody were injected at 0, 200, 400, 600, and 800 s. Both systems delivered similar results.

Reduce costs with regenerative Strep-Tactin®XT biosensor chips

An interaction analysis always includes several measurements. Therefore, the option of reusing a biosensor chip would be highly beneficial since it reduces the costs associated with an experiment.

The possibility for biosensor chip regeneration exists for both the Avi-tag/streptavidin and the Twin-Strep-tag®/Strep-Tactin®XT system. However, important differences between these two systems are effectiveness, convenience of the procedure and costs for regenerating a chip.

Due to its high affinity, the Avi-tag/streptavidin interaction is too strong to dissolve and therefore the only efficient way to reuse a chip is to remove the whole ligand/streptavidin complex.

A new generation of streptavidin biosensor chips come with an established regeneration protocol. In this protocol, streptavidin is released during regeneration and must be re-applied before the next ligand application. A disadvantage of these streptavidin biosensor chips is that they are twice as expensive as Strep-Tactin®XT biosensor chips. A cheaper, but complex and often inefficient way is to use conventional streptavidin biosensor chips. For the regeneration of these chips, optimal regeneration conditions must be determined for each different ligand. After all, a new chip may be needed for each measurement anyway, since the procedure might also affect the ligand’s activity.

In contrast to Avi-tag/streptavidin, which is a nearly irreversible binding, the Twin-Strep-tag®/Strep-Tactin® interaction can be dissolved easily. Although the binding affinity is very high, the addition of guanidine hydrochloride (GuHCl) causes the complete release of the strep-tagged ligand from Strep-Tactin®XT. Strep-Tactin®XT remains on the biosensor chip and is unaffected by this procedure. The protocol is fast and simple and allows regenerating the chips more than 30 times without adverse effects on functionality.

Stable performance of Strep-Tactin®XT biosensor chips after several regeneration cycle

Fig. 4: Ligand capture level of CD45-Twin-Strep-tag® on a Strep-Tactin®XT coated CM5 chip after several regeneration cycles. Regeneration was performed with three consecutive 1-minute injections of 3 M GuHCl.

A superior alternative to His-tag/NTA – Stable, reversible and highly specific immobilization on SPR sensor chips using Strep-Tactin®XT

Xantec bioanalytics teamed up with IBA Lifesciences to introduce Strep-Tactin®XT/Twin-Strep-tag® system as a new derivative on SPR sensor chips.

The Strep-Tactin®XT/Twin-Strep-tag® system offers several key benefits over the traditional His-tag/NTA system for SPR sensor chips, including high affinity and binding stability in the low picomolar range, making it exceptionally reliable for protein immobilization. The regeneration process is straightforward, supporting over 100 cycles with minimal capacity loss, which enhances the system’s usability and efficiency.

Additionally, the Strep-Tactin®XT/Twin-Strep-tag® system demonstrates significantly reduced nonspecific interactions compared to the His-tag/NTA system, particularly benefiting applications involving larger analytes. Immobilization is site-specific and occurs under physiological conditions, preserving high biological activity and ensuring reproducibility.

The Strep-Tactin®XT/Twin-Strep-tag® sensor chip variants are optimized for different applications such as protein-protein interactions, fragment screening, and concentration analyses. Overall, the system provides a more stable, specific, and efficient method for SPR sensor chip immobilization, addressing the limitations of the His-tag/NTA system and enhancing the reliability of SPR-based analyses.

Application examples

Strep-Tactin®XT

Strep-Tactin®XT binds with high affinity Twin-Strep-tag® proteins, making it perfectly suitable for SPR analysis. The examples show the coupling of Strep-Tactin®XT on different SPR chips and possible regeneration conditions for re-use of Strep-Tactin®XT-coated chips.

Coupling on CM5

We recommend to use the following conditions for optimal Strep-Tactin®XT coupling:

• 20-50 µg/ml Strep-Tactin®XT
• max. 20 mM acetate, pH 4.5

Using these conditions for Strep-Tactin®XT coupling, efficient and reproducible coupling results can be obtained.

Coupling on CM4

The membrane protein CB2 was fused with Twin-Strep-tag® and then immobilized on a CM4 sensor chip coated with Strep-Tactin®XT.

Used conditions:

• 50 mM Tris pH 7.5, 100 mM NaCl
• 10 % glycerol, 0.1 % DDM, 0.5 % CHAPS, 0.1 % CHS

Data was published in A. Yeliseev et al., Protein Expression and Purification 131 (2017) 109-118

Regeneration

Three different regeneration conditions were tested on Strep-Tactin®XT CM5 sensorchips:

• 10 mM NaOH / 500 mM NaCl
• 3 M MgCl2
• 3 M GuHCl

for three different Twin-Strep-tag® fusion proteins:

• mCherry-Twin-Strep-tag
• GAPDH-Twin-Strep-tag
• mAb-Twin-Strep-tag

We recommend the application of 3 M GuHCl for optimal regeneration conditions, since this buffer led to the best regeneration results for all test proteins. However, depending on the target protein, other buffers are applicable as well.

StrepMAB-Immo

Due to the nearly irreversible binding of Strep-tag®II proteins, StrepMAB-Immo can be used for SPR analysis. The example shows the stable capture of a Strep-tag®II fusion protein on a SPR chip coated with StrepMAB-Immo.

Chips for SPR were coated either with StrepMAB-Immo or with a competitive antibody (Competitor Q). Subsequently, a Strep-tag®II fusion protein was captured, and the binding stability was determined using a Biacore 3000.

During the washing phase, the recombinant Strep-tag® protein of interest remains tightly bound to StrepMAB-Immo, while a significant amount of Strep-tag® protein is washed off using the competitive antibody.

Bio-layer interferometry

Bio-layer interferometry (BLI) is like SPR a label-free optical biosensing technology for analyzing biomolecular interactions, e.g., antigen-antibody interactions, in real-time and allows quantification of their binding strength and kinetics. For BLI experiments, thin needles, called biosensors, are coupled at the tip with a desired ligand and dipped into the sample to capture the analyte. To measure whether the analyte is bound to the ligand, white light is passed through the biosensor and the reflected light from the tip surface is measured. If the ligand interacts with the analyte, the layer on the surface of the tip is thicker compared to the ligand alone, which leads to a different wavelength that is reflected. The BLI instrument (Octet® or Gator®) measures both the reflected light from the ligand alone and the ligand-analyte complex. The wavelength shift between both reflection patterns creates an interference pattern, which is subsequently used for calculation of binding strength and kinetics.

But what is the best way to bind the ligand to the surface of the biosensor? In principle, the ligand can be bound directly via chemical coupling methods like EDC/s-NHS coupling. But this needs time to find the optimal reaction conditions and can reduce the activity of the ligand. In addition, the orientation of the ligand can vary, which can reduce the accessibility for the analyte and can lead to inconsistent measurement results between the biosensors.

These disadvantages can be prevented if an indirect binding via the Strep-tag® technology is used. Instead of the ligand, Strep-Tactin®XT is first immobilized on the biosensor and the ligand tagged with the Twin-Strep-tag® is captured afterwards by it. Since the tag is at a defined position, all proteins (ligand) captured by Strep-Tactin®XT will be presented in the same orientation. Furthermore, the pM affinity of Strep-Tactin®XT for the Twin-Strep-tag® allows specific capture of Twin-Strep-tagged fusion proteins from complex samples, e.g., culture supernatants, leading to reliable and robust data. All reagents necessary for fast and simple coating of Strep-Tactin®XT biosensors are provided in our Strep-Tactin®XT BLI Coupling Kit and ready-to-use Strep-Tactin®XT biosensors are provided by Gator®.

This application note demonstrates how the Strep-Tactin®XT technology can be used in combination with BLI and SPR analytic devices.

Bio-layer interferometry (BLI) in combination with Strep-Tactin®XT for uniform and specific capture of Twin-Strep-tag® fusion proteins.

Quantification and kinetic analysis of Twin-Strep-tag® fusion proteins

Twin-Strep-tag® is widely utilized for purifying recombinant proteins in fields such as structural biology, enzyme engineering, and protein characterization. Its highly specific binding properties enable efficient purification of target proteins with high purity. The Twin-Strep-tag® is particularly advantageous compared to the His-tag for proteins that need to be purified at physiological pH in order to maintain their functionality.

Webinar

This webinar describes how Strep-Tactin®XT can be used for efficient purification of nanobodies produced in mammalian cell lines and how Strep-Tactin®XT biosensors can be used to analyze the nanobodies using BLI.

ELISA – Enzyme-linked immunosorbent assay

Direct ELISA

Indirect ELISA

Sandwich ELISA

The enzyme-linked immunosorbent assay, short ELISA, is a commonly used method for detection and quantification of ligands, such as proteins, peptides, or hormones even in a complex mixture. ELISA is a plate-based technique in which, depending on the type, either the target ligand or the capturing reagent is immobilized on a microplate. While there are different types of ELISA, the essential components are always consistent. Firstly, a ligand of interest, which is to be detected and potentially quantified. Secondly, an antibody, like StrepMAB-Immo or StrepMAB-Classic, or other detection reagent, like our conjugated Strep-Tactin®XT for specific recognition of the ligand. Lastly, an enzymatic reporter, the substrate conversion of which can be used as measurable signal.

While different formats exist, the basic principle when performing any kind of ELISA is the same.

1. Coating or capturing of the directly or indirectly immobilized ligand on the microplate.
2. Blocking of free surface-binding sites of the microplate.
3. Detection of the ligand by using ligand-specific reagents, e.g. antibodies.
4. Readout of signal produced by the enzymatic reaction of labeled primary or secondary antibody.

Depending on the experimental needs, one can choose between direct, indirect and sandwich ELISA. The formats differ in the way of capturing and detecting the target ligand.

The direct and indirect ELISA both start with the immobilization of the ligand on a microplate. Whereas the direct ELISA only requires a labeled primary antibody for detection, in an indirect ELISA an unlabeled primary antibody first, and then a labeled secondary antibody is used. However, instead of a primary antibody it is possible to use Strep-Tactin® or Strep-Tactin®XT conjugates for Strep-tagged ligands, which is particularly beneficial because a second antibody for sensitive signal detection becomes redundant. Alternatively, StrepMAB-Classic conjugated with HRP can be applied, also avoiding the use of a secondary antibody.
In a sandwich ELISA, the plate is coated with a capturing molecule, like an antibody. Subsequently, the ligand is added and “sandwiched” between the capturing and the detecting antibody. Here, the primary antibody detecting the ligand is generally unlabeled, while a labeled secondary antibody is added for quantification. For capturing a desired Strep-tagged protein, plates can be coated using our StrepMAB-Immo or StrepMAB-Classic antibody. However, we recommend StrepMAB-Immo for this purpose due to its higher affinity towards the Strep-tag®. Convenient pre-coated plates with Strep-Tactin® or Strep-Tactin®XT are additionally available.
As enzymatic reporter, horse-radish peroxidase (HRP) is most commonly applied. It convert a certain substrate into a detectable product. As substrate, a variety of different chromogenic, fluorescent, and chemiluminescent products are available, always depending on the desired sensitivity and the laboratory equipment at-hand. In case a fluorescent detection method is desired, we offer various fluorescent labels conjugated to Strep-Tactin®XT, StrepMAB-Immo or StrepMAB-Classic.

The western blot technique is an expedient method for the identification of specific proteins from a complex mixture. After extraction of the protein mixture, e.g. via cell lysis, the first step is to separate the proteins based on their molecular weight using gel electrophoresis. For protein separation by size, usually a polyacrylamide gel is prepared. Next, the proteins are transferred or blotted from the gel to a solid support, generally a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. Because of its efficiency and speed, electrophoretic transfer is the most common blotting procedure. The gel and the membrane are “sandwiched” between two electrodes and the proteins are transferred due to their electrophoretic mobility, influenced by factors such as charge and size.

Since the membranes have a high affinity for proteins, the remaining binding sites of the surface need to be blocked after the transfer to prevent unspecific binding of the detection antibodies. A variety of different buffers and reagents is used in the whole process and, to minimize the signal-to-noise ratio, sufficient washing is required as intermediate step.

For detection of the antigen, it can be chosen between direct or indirect detection. For direct detection of Strep-tagged proteins, only a single labeled and target-specific antibody, such as our StrepMAB-Classic conjugated to HRP, is required. While it is quite common to use antibodies for detection, our Strep-Tactin® products conjugated to HRP or AP represent an equally beneficial alternative. These products are based on our Strep-tag® technology.

The combination of a primary antibody detecting the target and a labeled secondary antibody is widely used as well, as the second antibody leads to a signal amplification and provides different options for multiple detection methods. The choice of primary and secondary antibody depends on various parameters, such as the origin of the target protein, the species of the primary antibody or the desired detection method. When working with Strep-tagged proteins, our murine StrepMAB-Classic antibody is a good choice as primary antibody.

Nowadays widely applied labels are fluorophores or enzymes. As in the case of ELISA, horse-radish peroxidase (HRP) or alkaline phosphatase (AP) are the most common enzymes. Even though enzymatic labels require extra steps and usually have to be optimized, they are used most extensively due to the high sensitivity and the flexibility in detection when choosing between chromogenic, fluorogenic, and chemiluminescent substrates. When using fluorophores as labels, fewer steps are required, since no substrate development is necessary. For this purpose, we offer various fluorescent labels conjugated to Strep-Tactin®XT, StrepMAB-Immo or StrepMAB-Classic. The choice of the experimental procedure is generally dependent on the laboratory equipment, e.g., special devices for the detection of a fluorescent signal are needed.

Altogether, the relatively easy protocol for western blots offers a broad range of potential utilizations, such as detecting post-translational-modifications or verifying protein cloning, and makes it such a popular application.

Western blot using Strep-Tactin® conjugated AP

Horse-radish peroxidase conjugated to Strep-Tactin®
Western blot using Strep-Tactin® conjugated HRP

Horse-radish peroxidase conjugated to StrepMAB-Classic
Western blot using StrepMAB-Classic conjugated HRP

Application examples

Protein detection with Strep-Tactin® Conjugates

Strep-Tactin® conjugates can be used for several assay applications. As example, the application of Strep-Tactin®HRP and Strep-Tactin®AP for Western Blot as well as Dot Blot is shown.

Strep-Tactin®HRP

Application: Western Blot

1-1000 ng/lane of GFP C-terminally tagged with Strep-tag®II (28 kDa) were separated by SDS-PAGE and blotted onto a membrane. Detection occurred with Strep-Tactin® HRP.

Application: Dot Blot

0.5-500 ng/spot of GFP-Strep-tagII (28 kDa) or alkaline phosphatase C-terminally tagged with Strep-tag®II (monomer 48.5 kDa) were spotted onto a membrane. Detection occurred with Strep-Tactin® HRP.

Strep-Tactin®AP

Application: Western Blot

1-1000 ng/lane of GFP C-terminally tagged with Strep-tag®II (28 kDa) were separated by SDS-PAGE and blotted onto a membrane. Detection occurred with Strep-Tactin® AP.

Application: Dot Blot

0.5-500 ng/spot of GFP-Strep-tagII (28 kDa) or alkaline phosphatase C-terminally tagged with Strep-tag®II (monomer 48.5 kDa) were spotted onto a membrane. Detection occurred with Strep-Tactin® AP.

Immunohistochemistry and immunocytochemistry

Amongst the immunostaining methods, immunohistochemistry is the most prominent one, in which whole tissues can be visualized, and thus, specific targets can be detected. In contrast to immunohistochemistry, in immunocytochemistry the focus is not on tissues but on cells. The advantage of both techniques is the possibility of imaging proteins in their proper histological context, which enables the localization of cellular compartments and can be detected using fluorescence microscopy. In both methods the sample fixation is essential to preserve the morphology and structure.

While both, chromogenic or fluorescent labels are utilized for detection, fluorescent labeling is especially popular due to the possibility of multicolor staining of different targets. StrepMAB-Classic or StrepMAB-Immo conjugated to different dyes can be applied for direct labeling of Strep-tagged targets. As viable alternative to antibodies, Strep-Tactin® and Strep-Tactin® XT with different fluorescent conjugates can be used as well. Due to their strong and highly specific interaction with Strep-tagged targets, both generate signals with high sensitivity, avoiding the need for a secondary antibody. However, when only small amounts of Strep-tagged protein are present, the unconjugated murine antibodies StrepMAB-Classic and StrepMAB-Immo can be used combined with a secondary antibody of choice.

Immunohistochemistry/-cytochemistry using fluorescently labeled Strep-Tactin®

Immunohistochemistry/-cytochemistry using fluorescently labeled StrepMAB antibody

StrepMAB-Classic

Application: Confocal microscopy (Immunofluorescence)

Dilution: 1:500

HeLa cells were grown on glass coverslips, fixed (4% PFA/PBS), quenched (15 mM glycine/PBS), permeabilized (0.1% saponin/PBS), blocked (1% BSA, 0.01% saponin in PBS), and stained with StrepMAB-Classic (1:500) to confirm the expression of SARS-CoV-2 ORF3a-Twin-Strep-tag. Anti-mouse 488 was used as secondary antibody. Nuclei were stained with DAPI and the imaging occurred with a LSM700 confocal microscope (63×/1.4 NA oil immersion objective; ZEISS). Source: Data kindly provided from James Edgar, Department of Pathology at the University of Cambridge.

Application example

Flow cytometry

Fluorescently stained cells are frequently deployed in flow cytometry analysis, which can be conducted both, before and after cell isolation, delivering insights into cell functions for basic research and clinical trials. Here, the labeled cells pass a light beam inside a flow cytometer with high velocity, thereby providing a multitude of information on physical and chemical properties and a lucid quantitative display of different cell populations. Fluorescence-activated cell sorting (FACS) using a sorter is a specialized form of flow cytometry, where target cells are not subsequently discarded, but rather rigorously divided into defined subpopulations according to their fluorophore label – hence, surface marker identities – and retained for further downstream processing.

The Strep-tag® system can be used to stain and sort a cell of interest. One option is the detection of a surface protein fused to a Strep-tag®II or Twin-Strep-tag® via fluorescent Strep-Tactin® or Strep-Tactin®XT conjugates. Alternatively, fluorescently conjugated StrepMAB-Immo antibodies can be used as well.

Another option is the staining and sorting of cells via a strep-tagged protein. In this case, the protein of choice, which binds the cell, must be combined with Strep-Tactin® or Strep-Tactin®XT conjugates. This leads to the generation of a staining reagent suitable for flow cytometry. This cell selection approach is commonly used for e.g. the selection of antigen-specific B or T cells, which are targeted by specific antigens or peptide-loaded MHC class I molecules, respectively.

Application examples

Detection with Strep-tag® Antibodies

StrepMAB-Classic and StrepMAB-Immo can be used for detection of Strep-tag®II or Twin-Strep-tag® proteins on cell surfaces, within the cell, in cell lysates, or in eluates after protein purification. As example, the application for flow cytometry is shown.

StrepMAB-Classic DY-549

Application: Flow cytometry
Dilution: 1:500

HeLa cells were transiently transfected with the coding sequence of SARS-CoV-2 ORF3a-Twin-Strep-tag. After 48 hours transfection, cells were detached, gently trypsinised and stained with StrepMAB-Classic DY-549 (1:500, 0.5% BSA + 1 mM EDTA, 30 min on ice). Intracellular SARS-CoV-2 ORF3a-Twin-Strep-tag levels were measured by flow cytometry (4 laser Cytoflex S, Beckman Coulter) Source: Data kindly provided from James Edgar, Department of Pathology at the University of Cambridge.

Fluorescent cell staining for flow cytometry with Strep-Tactin® and Strep-Tactin®XT conjugates

The Strep-tag® technology provides different options for cell staining.

Direct vs. indirect staining

Direct staining of cells expressing a protein with a Strep-tag®II or a Twin-Strep-tag® on the surface

Indirect staining via a cell-binding protein (e.g. a Fab fragment, a MHC molecule or an antigen) that is fused to a Strep-tag®II or a Twin-Strep-tag®

Direct staining

The Strep-tag®II or Twin-Strep-tag® are short peptide sequences that can be expressed on a cell surface without affecting the properties of the cell. Fusing one of the tags to a surface protein of choice enables easy selection of cells that successfully express this protein. MEXi cells expressing a surface protein fused to a Twin-Strep-tag® were mixed with native MEXi cells at a ratio of approximately 1:1. 1 x 106 total cells were stained with Strep-Tactin®XT DY-649 at a dilution of 1:5000* (A) or with Strep-Tactin®XT APC at a dilution of 1:1000* (B). Staining was performed in 100 µl Buffer CI (1x PBS with 1 mM EDTA and 0,5% BSA) for 30 min at 4°C.

*Titration of optimal dilutions might be required for individual experimental setups. In addition, the choice of conjugate depends on factors such as protein density on the cell surface. Due to their brighter staining, PE and APC conjugates can be advantageous for low expressed targets.

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Indirect staining via strep-tagged protein (Fab)

For cytotoxic T cell staining, 200 ng anti-human CD8 Fab fragment (50 kDa) fused to a Twin-Strep-tag® was pre-incubated with 75 ng of Strep-Tactin®XT APC (A) or Strep-Tactin®XT DY-649 (B) for 10 min at 4 °C. The formed complexes were added to 5 x 106 peripheral blood mononuclear cells (PBMCs) and incubated for 20 min at 4 °C in the dark. Afterwards, additional staining antibodies were added. Both fluorescent conjugates achieved clear separation of positive and negative cell populations, demonstrating a flexible choice of fluorophores for highly expressed target proteins such as CD8 on the surface of T cells.

Indirect staining of T cells via strep-tagged protein (MHC)

For staining low affinity targets such as T cell receptors, multimerization of the ligand is required to enable stable binding of the detection reagent to the cell. This multimerization can be achieved with Strep-Tactin® or Strep-Tactin®XT PE or APC conjugates.

For antigen specific T cell staining, 200 ng of an MHC class I molecule fused to a Twin-Strep-tag® (50 kDa) refolded with a cytomegalovirus (CMV)-derived peptide was pre-incubated with 75 ng of Strep-Tactin® PE (A) or Strep-Tactin®XT PE (B) for 15 min at 4 °C. 1 x 107 PBMCs in 100 µl buffer were added to the staining complexes and incubated for 20 min at 4 °C. Afterwards, additional staining antibodies were added.

Strep-Tactin®XT PE achieved a brighter staining intensity than Strep-Tactin® PE, indicating that Strep-Tactin®XT PE and APC are the preferred conjugates for low affinity and low expressed targets such as specific T cell receptors. Strep-Tactin®XT DY-649, DY-549 and DY-488 conjugates are not recommended for this approach, since their lesser brightness does not achieve sufficient staining (data not shown).

Investigation of Protein-Protein Interactions with the help of the Strep-tag® technology

Understanding protein-protein interactions (PPIs) is fundamental to the field of molecular biology. These interactions regulate crucial biological processes, from signal transduction and immune responses to maintaining cellular structure and function. Many diseases are linked to protein interactions. By investigating these interactions, researchers can identify potential therapeutic targets, understand disease mechanisms, optimize leads during the drug discovery process, or discover biomarkers for disease diagnosis and monitoring.

The key steps to identify and characterize the interaction between two specific proteins include:

• Experimental Design: Define the target protein and choose the appropriate methods based on the specific goals and available resources
• Lead Identification: Collect data to identify potential interaction partners. This step involves screening and identifying promising leads that show significant interaction.
• Confirmation: Validate initial findings using complementary techniques in-vitro or in-vivo.
• Characterization: Further characterize the interaction structurally and functionally, for example by measuring binding kinetics in SPR

Figure 1: Typical workflow of an experiment to identify and analyze protein-protein interactions

Experimental Design: Selecting experimental conditions

To ensure an easy workflow, as well as accurate and reliable results, several factors should be considered while planning the experimental setup. Selecting the appropriate detection method, protein expression system and conditions for sample preparation are vital. Incorporating protein tags, such as Twin-Strep-tag®, His-tag or FLAG-tag can offer advantages.

Protein tags facilitate the purification and detection of target proteins and can improve the efficiency of the experiment. They can also aid in further steps, like the ability to immobilize the protein during analysis steps, for example in SPR. To offer benefits to the analysis workflow, the ideal tag should have the following properties:

• No unspecific interaction
• High affinity
• Little to no influence on protein structure
• Small size
• Reversible binding
• Multiple possible applications

Among the most commonly used tags, the Twin-Strep-tag® possesses all these properties and offers the greatest benefit for PPI analysis.

Lead identification: Affinity chromatography, mass spectrometry and bioinformatics

After a target protein has been chosen, the possible interaction partners have to be identified. Isolating the target protein from the cell while keeping its interactions intact is a convenient way to do this. Pull-down assays and Co-immunoprecipitation (Co-IP) are two effective methods for detecting protein-protein interactions. Co-IP utilizes immobilized antibodies to purify the bait protein along with the prey from cell lysates, enabling the analysis of the entire protein complex. In contrast, pull-down assays involve fusing the target protein to an affinity tag and capturing it, along with any bound proteins, using a corresponding immobilized ligand.

Figure 2: Differences between Co-IP via protein specific antibodies and a pull-down, which is performed via a specific protein tag.

While antibodies are a great choice when specific ones antibodies are available, this is not always the case. In addition, specificity and binding affinity can vary between different antibodies, leading to unreliable results. Pull-down assays, for example via Twin-Strep-tag® and Strep-Tactin®XT offer several advantages. The same tag and ligand system can be used for different proteins, while the same affinity and specificity is guaranteed. Furthermore, they offer mild elution conditions, which preserves the interaction between the bait and prey proteins.

In a subsequent step, the potential interaction partners are then identified, for example by mass spectrometry. These experimental methods are complemented by bioinformatic methods, predicting potential interaction partners based on sequence and structural data from databases. Combining computational and experimental procedures enhances the accuracy and efficiency of lead identification, allowing researchers to narrow down the pool of candidates that need to be screened. Once the most promising leads are identified, each interaction must be individually studied for detailed characterization to understand the biological significance and mechanism of the interactions.

Figure 3: Using a pull-down-assay to identify protein interactions: The tagged target protein is expressed in the host cell, after which it is purified under mild conditions via the Twin-Strep-tag®. Prey proteins that were co purified are separated from the bait protein in gel electrophoresis, digested and then identified in mass spectrometry.

Confirming the interaction

After potential interaction partners have been identified, further steps are required to confirm the interaction. For this, the original bait protein and the prey proteins are recombinantly produced with modifications that facilitate the detection of their interaction.

One approach involves labeling the proteins with agents that generate a signal when brought into close proximity by the interaction. These agents can be parts of proteins or specific fluorophores. For example, in Homogeneous Time-Resolved Fluorescence (HTRF) assays, donor and acceptor fluorophores are conjugated to molecules that recognize the binding partners, such as Strep-Tactin® or antibodies. This assay relies on Förster resonance energy transfer (FRET), where energy is transferred from a donor fluorophore to an acceptor fluorophore and induces a signal when they are close together. HTRF offers high sensitivity and reduced background noise through time-resolved measurement

Figure 4: Principle of HTRF for confirmation of protein interactions. The donor fluorophore is conjugated to an antibody specific to the prey protein, while the acceptor fluorophore is conjugated to a ligand that binds to the tag of the bait protein. The donor fluorophore is excited by a light of a certain wavelength and transfers its energy to the acceptor fluorophore if they are in close proximity, which results in a measurable signal.

Figure 5: Confirmation of protein interaction in SDS page after Pull-down-assay. Prior to the experiment, the tagged bait protein, prey protein and an unrelated control protein are recombinantly expressed. The proteins are then mixed in different combinations and purified via the tag: The bait protein with the control protein, the bait protein with the prey protein and the prey protein on its own. If the prey protein is only visible in the gel if it was mixed with the bait protein, the interaction is confirmed.

Another straightforward method, especially if the initial screening was performed via a pull-down assay, is a subsequent pull-down. In this method, the chosen prey protein is recombinantly expressed and mixed with the bait protein. An affinity purification targeting the bait protein is then conducted. The presence of the prey protein is confirmed by Western blot analysis, comparing results to negative controls to validate the interaction. Other methods not only confirm the interaction but also provide additional insights. For example, protein crystallography can determine protein structure, or Surface Plasmon Resonance (SPR) can measure binding kinetics. These advanced techniques offer detailed information on the nature and dynamics of the interaction.

Characterizing the interaction: SPR and BLI

Once an interaction partner has been identified and confirmed, the interaction can be further characterized to gain deeper insights into its biological function. Factors such as binding kinetics and affinity are crucial for understanding the role of the interaction in the organism. Techniques like surface plasmon resonance (SPR) and biolayer interferometry (BLI) are particularly useful for this purpose.

Both SPR and BLI offer real-time analysis of protein interactions, allowing for precise measurement of association and dissociation rates. Both techniques require the immobilization of the bait protein on a sensor or chip surface in a stable and oriented manner. The Twin-Strep-tag® technology is highly advantageous for this step due to its picomolar affinity, ensuring secure and directional immobilization of the bait protein. This allows the measurement of interactions of low to high affinites. Moreover, surfaces coated with Strep-Tactin®XT can be regenerated and reused multiple times, making these biosensor platforms cost-effective.

By utilizing the strengths of SPR and BLI, along with the robust immobilization provided by affinity tags like the Twin-Strep-tag®, researchers can obtain detailed kinetic and affinity data. This information is essential for revealing the functional roles of protein-protein interactions and their potential implications in cellular processes and disease mechanisms.

Figure 6: Measuring Binding kinetics via SPR: The tagged bait protein can bind to the SPR chip via the high affinity Twin-Strep-tag® to Strep-Tactin®XT. The prey protein is applied to the chip for the measurement. The resulting binding kintetics are essential information to characterize an interaction between two proteins.

References

• Gordon, D.E., Jang, G.M., Bouhaddou, M. et al. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature 583, 459–468 (2020). https://doi.org/10.1038/s41586-020-2286-9
• He L, Valignat MP, Zhang L, Gelard L, Zhang F, Le Guen V, Audebert S, Camoin L, Fossum E, Bogen B, Wang H, Henri S, Roncagalli R, Theodoly O, Liang Y, Malissen M, Malissen B. ARHGAP45 controls naïve T- and B-cell entry into lymph nodes and T-cell progenitor thymus seeding. EMBO Rep. 2021 Apr 7;22(4):e52196. https://doi.org/10.15252/embr.202052196
• Hou YN, Cai Y, Li WH, He WM, Zhao ZY, Zhu WJ, Wang Q, Mai X, Liu J, Lee HC, Stjepanovic G, Zhang H, Zhao YJ. A conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1. Nat Commun. 2022 Dec 22;13(1):7898.
  https://doi.org/10.1038/s41467-022-35581-y

Products

Monoclonal antibody development & production

The identification and production of monoclonal antibodies is essential for investigating immune responses and developing therapies. In addition, they can be utilized for the direct and indirect detection of antigens in various assays, as capture molecule immobilized on microplates, or as ligand for protein purification, making them a versatile tool in biotechnology.

Different methods were developed for monoclonal antibody discovery including hybridoma technology, phage display or single B cell sorting.

Their common aim is the identification of high affinity antibodies that recognize a specific antigen of choice. This makes the antigen a central component for antibody development. Fusing a Twin-Strep-tag® to an antigen helps to make this central component applicable for various steps in the development process.

The Twin-Strep-tag® suitable for:

• Antigen (protein) purification and analysis
• Immunization
• Antigen-specific B cell staining and isolation via FACS
• ELISA and Western blot
• Biosensor applications
• Biopanning

This way, the Strep-tag® technology can centralize various steps onto one platform, making antibody development time-saving and cost-efficient.

Figure 1: An antigen fused to a Twin-Strep-tag® as central component for all applications needed in antibody development (e.g. with hybridoma technology, single B cell sorting or phage display)

Antigen expression & purification for antibody development

Antigen expression & purification for antibody development

The production of an antigen is a key process in antibody development. It is needed for several steps in the whole procedure such as immunization, screenings and antibody characterizations.

The advantages of using the Strep-tag® system for the antigen purification process are:

• Highly pure antigen preparations suitable for immunization and further downstream processes
• Straightforward isolation procedure without tedious optimization steps
• Reusability even after CIP (Clean-in-place) procedures

Figure 3: Different Strep-Tactin® conjugates allow for analysis methods such as western blot or ELISA.

Immunization for antibody development

The purification of proteins with the Strep-tag® technology results in a highly pure antigen that is suitable for immunizing a host species. Additional optional processing steps include the removal of biotin, the eluent, from the elution fraction via dialysis or size exclusion and/or TEV cleavage of the Twin-Strep-tag®. However, since the tag has a small size of only 28 amino acids, the immunogenicity is expected to be low.

Figure 4: The Twin-Strep-tag® is a short peptide, therefore the removal prior to immunization is not strictly necessary. Optionally, the tag can be removed via a TEV protease.

Antigen-specific B cell staining & sorting for antibody development

For some antibody development approaches, antigen-specific B cells are isolated from blood or other tissues of seropositive individuals, which encountered the antigen by infection or immunization. Here one can make use of the antigen fused to the Twin-Strep-tag® by incubating it with:

Fluorescent Strep-Tactin® or Strep-Tactin®XT conjugates or
Strep-Tactin® Magnetic Microbeads or
Strep-Tactin® TACS Agarose
This generates selection reagents that are suitable for the staining and sorting of antigen-specific B cells via flow cytometry, magnetic or affinity chromatographic isolation, respectively.

B cells that recognize the antigen of choice are selected from e.g. peripheral blood mononuclear cells (PBMCs). Single cells can be sorted directly into lysis buffer for antibody sequencing or taken into culture for antibody production. This method for monoclonal antibody production offers the possibility to extract B cells and consequently also antibody sequences directly from humans, thereby enhancing the chance to identify antibodies that are readily suitable for therapeutic purposes.

Figure 5: Via its tag, the antigen can be combined with Strep-Tactin® conjugates for e.g. fluorescent staining and sorting of B cells.

Example publications for antigen-specific B cell staining & sorting

Clone/antibody screening & antibody characterization for antibody development

Independent from the method chosen for monoclonal antibody discovery, screening steps are key to finally identify the best performing antibody.

Antibody purification

After the best antibody was identified for a specific antigen, it has to be expressed in e.g. a cell culture system. The produced antibodies subsequently have to be extracted from cell lysates, cell culture supernatants, or biological probes. The purification can take place without affinity tag via the physicochemical properties, antigen-specific affinity or the antibody class. Physicochemical properties are the molecular weight, charge, or clusters of specific residues.

Depending on the origin of the sample, purification with the help of physicochemical properties or antigen-specific affinity can lead to the isolation of further proteins and antibodies with similar properties besides the target antibody. Thus, if a specific antibody with a high purity should be obtained, antibody class-specific affinity chromatography is recommended, such as Protein A, which can bind immunoglobulins (antibodies) within the Fc region of their heavy chain without regard to antigen specificity.

Choosing an affinity tag-based purification procedure is also possible and can be beneficial, especially if the antibody is to be implemented in subsequent immobilization or detection. For this purpose, the application of the Strep-tag® technology is highly recommended. The antibody can simply be isolated in high purity with a resin perfectly suitable for large proteins, Strep-Tactin®XT 4Flow®. The different formats available for protein purification make it a very flexible system that allows for individual adaptations of sample number and size.

Figure 8: Due to the picomolar affinity of Strep-Tactin®XT to the Twin-Strep-tag®, biosensor applications such as surface plasmon resonance (SPR) are possible.

SPR & BLI

The antibodies that are yielded by different antibody discovery and development approaches can be further characterized by biosensor applications such as surface plasmon resonance (SPR) or Bio-layer interferometry (BLI). Again, the Twin-Strep-tagged antigen is central in those analyses. Due to the picomolar affinity of the Twin-Strep-tag® to Strep-Tactin®XT, the antigen is immobilized sufficiently to allow the determination of binding kinetics to a selected antibody. This way, antibodies with the desired affinities are identified and chosen for large scale productions.

Figure 6: An antigen can be immobilized on Strep-Tactin®XT coated microplates via its Twin-Strep-tag® for antibody screening with ELISA. Due to the reusability of the plates, several screening rounds are possible.

ELISA

A Twin-Strep-tag® – antigen can be used for antibody screening by ELISA. Via its tag, it can be immobilized on Strep-Tactin®XT coated microplates. This way, the microplate is functionalized for capturing antibodies from e.g. B cell supernatants. Subsequent detection indicates which B cell clones produce specific antibodies. Usually, several selection rounds are required to find the best performing clone. Since Strep-Tactin®XT coated microplates can be regenerated several times, using the Strep-tag® technology in this process is cost saving and time efficient.

Biopanning

Monoclonal antibody discovery by phage display uses a library of antibody fragments that are expressed on the surface of bacteriophages. To find those bacteriophages that present antibody fragments that detect the antigen, a selection procedure called biopanning is utilized. For this method, the phages are exposed to an immobilized antigen. The ones that bind are eluted, amplified and used in a next selection round. For the selection rounds, Strep-Tactin®XT coated microplates with an immobilized Twin-Strep-tag® antigen offer a reusable, cost-saving system similar to antibody screening by ELISA. After the identification of antibody fragments that specifically bind the antigen, sequences have to be cloned into full length antibodies for further use in research or therapeutic applications. Since this step can potentially change the features of an antibody, additional characterization steps are necessary, making this method for monoclonal antibody discovery overall very labor-intensive.

Figure 7: Strep-Tactin®XT coated microplates are suitable for biopanning, a selection procedure employed in phage display. The plates can be regenerated several times, therefore multiple selection rounds are possible.

From protein purification to analytical applications with Strep-Tactin®XT

Strep-Tactin®XT is the high affinity variant for the purification of strep-tagged fusion proteins, providing binding affinities in the picomolar range for Twin-Strep-tag® while still maintaining binding reversibility and mild recovery of immobilized proteins. It is suitable for efficient protein purification independent of protein class and size, including challenging proteins as well as low abundant proteins.

Protein purification

Fused to magnetic beads, MagStrep® Strep-Tactin®XT, is the ideal tool for complex identification via pull-downs and fast small-scale batch purification in reaction tubes or 96-well plates.

In particular, Strep-Tactin®XT 4Flow® high capacity provides superior performance for the purification of strep-tagged proteins from diluted cell extracts and allows for intensive wash procedures with large volumes of wash buffer. Strep-Tactin®XT 4Flow® high capacity resin is stable over a wide range of pH and compatible with various buffer conditions, and can be reused at least 100 times without losing performance. Due to this frequent reusability, experimental costs can be significantly reduced.

Analytical applications

The near covalent affinity of Strep-Tactin®XT to Twin- Strep-tag® expands the range of use towards analytical applications such as high throughput screening, assay development and protein kinetic studies.

You can obtain Strep-Tactin®XT conjugated to microplates, fluorophores, or chips. Strep-Tactin®XT coated microplates ensure convenient diagnostic assays and high-throughput screenings with high stability and antibody-free protein immobilization. Moreover, immobilized biomolecules are presented to interaction partners in a uniform manner, which results in reliable and highly reproducible assay formats. The picomolar affinity is particularly valuable for surface plasmon resonance (SPR) analysis and bio-layer interferometry (BLI) and supports measurements with long dissociation times and slow off-rates. Moreover, Strep-Tactin®XT biosensors can be easily regenerated.

Key features of protein purification and analysis with Strep-Tactin®XT

• Highly selective binding properties leading to unparalleled protein purity (> 95 %)
• Exceptionally high affinity to Twin-Strep-tag® enables reliable use in analytic applications
• Bioactive target proteins due to rapid one-step purification under target specific conditions
• Variability in buffer conditions, e.g. high salts, detergents, metal ions, chelators or reducing agents
• Favorable for protein-protein interaction studies due to mild elution conditions that preserve natural protein structure
• Products covering the entire workflow from cloning, purification to analytical applications (ELISA, FACS, SPR, Microscopy, Western Blot, etc.)

Strep-Tactin®XT also ensures sharp elution profiles, achieving high concentration of the target protein. Strep-Tactin®XT even tolerates a wide pH range in addition to various detergents and additives, contributing to the universal usability of the system.

Strep-Tactin®XT purification cycle

Protein purification with Strep-Tactin®XT is very simple and fast, since the purification cycle for each target protein (Strep-tag®II or Twin-Strep-tag®) is the same. However, if a target protein needs a specific buffer composition, e.g. PBS, HEPES or no chelating agents, like EDTA, the buffers can be easily adapted without the need of changing the purification cycle. Strep-Tactin®XT resin accept various buffer compositions, reagents and additives.

Protein purification with Strep-Tactin®

Strep-Tactin® allows affinity purification of Strep-tag®II and Twin-Strep-tag® fusion proteins under physiological conditions. In contrast to other tags, these mild purification parameters preserve bioactivity of the protein and the high specificity between Strep-tag ®II and Strep-Tactin ® can yield over 99% purity after a single chromatographical step.

Strep-Tactin® is especially suitable for standard proteins with good expression rates and binding properties. Furthermore, Strep-Tactin® can be alternatively used for the purification of proteins where the binding to the high affinity variant Strep-Tactin®XT is too strong.

Strep-Tactin® purification cycle

Strep-Tactin® resin is used for the purification of Strep-tag®II or Twin-Strep-tag® proteins. Protein purification with Strep-Tactin® is straightforward and quick, as the purification cycle for each target protein remains consistent. Additionally, if a target protein requires a specific buffer composition, such as PBS, HEPES, or buffer free from chelating agents like EDTA, the buffers can be easily adapted without altering the purification cycle. Strep-Tactin® resin is compatible with various buffer compositions, reagents, and additives.

Unlock the potential of high-affinity purification with Strep-Tactin®XT

Strep-Tactin® and its high-affinity counterpart, Strep-Tactin®XT, are streptavidin mutants, which bind strep-tagged proteins reversibly. The differences lie in the binding strength of the possible tag-ligand combinations.
Strep-Tactin®XT stands out as the high-affinity option tailored for purifying strep-tagged fusion proteins. With binding affinities reaching the picomolar range for Twin-Strep-tag®, it retains reversible binding properties and ensures gentle recovery of immobilized proteins. This versatility extends to various protein classes and sizes, enabling efficient purification of challenging and low-abundance proteins alike. Strep-Tactin®XT fused to magnetic beads, MagStrep® Strep-Tactin®XT beads, is the ideal tool for fast small-scale batch purification in reaction tubes or 96-well plates, and protein complex identification via pull-downs.
In particular, Strep-Tactin®XT fused to 4Flow® agarose, Strep-Tactin®XT 4Flow® high capacity resin, offers exceptional efficacy in isolating strep-tagged proteins from diluted cell extracts, enabling thorough washes with substantial volumes of buffer. The binding affinity is stable across a broad pH spectrum and compatible with diverse buffer conditions, ensuring versatility. Moreover, the high stability of the resin ensures effective binding over long periods of time.

Higher protein yields with Strep-Tactin®XT

Several proteins tagged with Strep-tag®II or Twin-Strep-tag® were purified with Strep-Tactin® (ST HC) and Strep-Tactin®XT (STXT HC). In all examples, Strep-Tactin®XT shows the highest binding capacity. The observed binding capacities were 1.5 to 3.4-fold higher in experiments using diluted cell extracts, and 1.8-fold higher using concentrated extracts. Remarkably, lower protein concentrations have no significant influence on Strep-Tactin®XT total binding capacity. In contrast, a significantly decreased binding capacity is observed for diluted cell extracts with Strep-Tactin®.

Cost-effective protein purification

Strep-Tactin®XT resins are the most cost-effective resins due to their higher binding capacity. As compared to Strep-Tactin® resins, Strep-Tactin®XT resin yields more than 2-fold more protein per ml resin and costs up to 54% less per milligram of purified protein. Furthermore, Strep-Tactin®XT prevents the unwanted leakage of the target protein during the washing steps.

Most efficient binding: Strep-Tactin®XT:Twin-Strep-tag®

Strep-Tactin® and Strep-Tactin®XT were loaded with twin-strep-tagged red fluorescent protein mCherry. Strep-Tactin®XT binds the protein directly in the upper part (right column), while Strep-Tactin® resin binds over nearly the entire resin bed (left column). The lower affinity of the tag to Strep-Tactin® causes the protein to detach more easily, resulting in a lower binding capacity as the protein flows out of the column.

During the washing steps, the protein is tightly bound by Strep-Tactin®XT and no protein is lost during purification. In contrast, significant mCherry loss is observed with Strep-Tactin® resin. This result highlights that Strep-Tactin® resins are not compatible with large samples or washing volumes. In the purification steps, due to the lower affinity, the protein detaches and runs off the column as the column volume is increased.

High affinity of Strep-Tactin®XT enables SPR analysis and BLI

Strep-Tactin®XT can be efficiently immobilized on SPR and BLI sensors and enables capturing of TST-fused ligands with exceptionally high affinity in pM range. The system allows kinetic analyses of strong binding analytes with long dissociation times and thus overcomes the current limitations of other affinity tag-based capture systems, such as the His-tag. The possibility to capture diverse ligands even directly from culture media and a simple regeneration procedure of the biosensors add major value to the application of Strep-Tactin®XT in optical biosensor assays.

Read more about Strep-Tactin®XT technology in downstream applications in this application note. Also, find out why Twin-Strep-Tag® in combination with Strep-Tactin®XT is a better alternative to the commonly used Avi-tag in SPR here.

SPR chips were coated with Strep-Tactin® or Strep-Tactin®XT and used for immobilizing a CD45 nanobody tagged with Twin-Strep-tag®. The results show that immediately after capture, the nanobody dissociates from Strep-Tactin®. In contrast, with Strep-Tactin®XT the nanobody is bound stably over a long-period of time. These data demonstrate that Strep-Tactin®XT can be used to analyze high-affinity interactions with slow dissociation rates.

Convenient assays and high-throughput screenings with Strep-Tactin®XT

Strep-Tactin®XT coated microplates ensure convenient assays and high-throughput screenings for strep-tagged biomolecules. Especially, the combination Twin-Strep-tag®:Strep-Tactin®XT is highly stable with a T1/2 of 13 hours and affinity in low pM range. The tag-ligand combination is an efficient and elegant option for an antibody-free immobilization of proteins.

Moreover, the immobilized biomolecules are presented to interaction partners in a uniform manner, which results in reliable and highly reproducible assay formats. This minimizes non-specific binding concomitant with minimal coefficients of variation. Hence, the Strep-Tactin®XT coated microplates are a precise but cost-effective tool for high-throughput screenings and diagnostic assays.

Efficient immobilization of (Twin-)Strep-tag®II fusion proteins on Strep-Tactin®XT
(A) Schematic of the oriented binding of recombinant proteins with N-terminal or C-terminal Strep-tag® during an fluorometric microtiter plate (MTP) assay. (B) Using a fluorometric MTP assay the maximum binding capacity of Strep-Tactin®XT was examined and compared with Strep-Tactin®. Bacterial alkaline phosphatase (BAP, BAP-Strep-tag®II and BAP-Twin-Strep-tag®) was applied onto microplates each coated either with Strep-Tactin® or Strep-Tactin®XT. After washing, the amounts of bound BAP were determined demonstrating that Strep-Tactin®XT recovers significantly more protein. (C) Comparison of the relative maximal binding capacity showing the efficient immobilization of proteins on Strep-Tactin®XT.

References for Strep-Tactin^®XT

Protein purification

• The adaptive landscape of a metallo-enzyme is shaped by environment-dependent epistasis (Anderson, et al. (2021). Nature)
• CryoEM of RUVBL1–RUVBL2–ZNHIT2, a complex that interacts with pre-mRNA-processing-splicing factor 8 (Serna, et. al.(2022). Nucleic Acids Research)

High-throughput purification

• Affinity Purification of TST-Proteins using Strep-Tactin^®XT 4Flow^® high capacity in IMCStips^® (Cook, et al. (2022). Application Note)

SPR

• Application of Strep-Tactin^®XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor (Yeliseev, et al. (2017). Protein Expression and Purification)
• A conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1 (Hou, et al. (2022). Nature Communications)

Strep-Tactin®XT products

Specifications of Strep-tag® Protein Purification Resins

There are several resin versions available for the purification of strep-tagged target proteins. All resins differ in their properties and suitability for applications. Learn more about the different resin matrices in order to know when to use which resin.

Find out more about Strep-tag® protein purification

Here, we have grouped links to make it easier to find more information about Strep-tag® technology for protein purification. In our web shop, you can find the protein purification products that fit your research needs. Read the comprehensive comparison of the most used tag systems, Strep-tag® vs. His-tag. Browser through application examples and frequently asked questions about Strep-tag® purification system or download application notes in our download area.