Protein-Protein Interaction

Immunoprecipitation (IP), Co-immunoprecipitation (Co-IP) and pull-down assays represent different methods to concentrate the target from a mixture for further downstream analysis such as the abundance, the activation status or the protein-binding partners.

While IP is used for capturing one specific target from a mixture, Co-IP and pull-down assays not only focus on single proteins but also on molecules which are attached to the target protein. Identifying the cellular interaction partners of a protein of interest and determining the biological pathways involved is a necessary preliminary work to gain deeper insights into a target’s structure and function. When it comes to the investigation, IP, Co-IP and pull-down assays have become invaluable tools, which are built upon the affinity-based immobilization of a “bait” protein on a solid support, mostly magnetic or agarose beads.

The advantage of pull-down over IP and Co-IP are mild elution conditions. They retain the natural protein structure as well as protein complexes and prevent elution of unspecific bound proteins leading to robust results in subsequent analytic applications. This is especially the case for the Strep-tag^® technology.

For pull-down assays, the Strep-tag^® technology is compatible with magnetic and non-magnetic experimental setups as well as any protein class and offers versatile resins for the realization of a multitude of assays to detect interactions. For small amounts of a sample, MagStrep “type3” XT beads can be chosen or when up-scaling of the process is anticipated, agarose-based resins display a powerful option.

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.

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.

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.

Besides BLI and SPR, protein-protein interactions can be analyzed as well with the switchSENSE^® technology.

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