Protein to Protein Conjugation

Buccutite™ Crosslinking Technology

Figure 2. Direct ELISA curves were generated using the HRP-goat-anti-mouse IgG conjugate prepared using the Buccutite™ Peroxidase Antibody Labeling Kit and Lightning-Link® HRP Antibody Labeling Kit. Mouse monoclonal antibody (0-10 ng/well) was coated on a 96-well plate overnight, and then blocked with PBST and 2% BSA. Wells were washed and then incubated with 100 µL/well GAM IgG-HRP conjugate (100 ng/mL) for one hour. Wells were were washed again and then a TMB substrate solution (Cat No. 11012) was used to detect the immobilized mouse IgG with 30 minute incubation and read at 405 nm.

Table 1. Buccutite™ Peroxidase Antibody Labeling kits designed for labeling antibodies with HRP and poly-HRP.

Buccutite™ Rapid PE, APC, PerCP and Tandem Dye Antibody Labeling Kits

Phycobiliproteins, such as phycoerythrin (PE), allophycocyanin (APC) and peridinin-chlorophyll-protein (PerCP), have the capacity to generate fluorescence signals significantly brighter than any commercially available synthetic fluorophore. When conjugated to molecules having biological specificity, such as antibodies, protein A or streptavidin, phycobiliproteins yield extraordinarily luminescent probes, that have found considerable success in fluorescence imgaging applications that require high sensitivity but not photostability, primarily flow cytometry, FACS and immunophenotyping. Moreover, phycobiliproteins can be labeled with small organic fluorophores to generate tandem dyes with exceptionally long Stoke’s Shifts for multiparametric analysis using a single excitation source. For example, FITC, PE, PE-Cy5 and PE-Cy7 are ideal for multiparametric analysis since all four fluorophores can simultaneously be excited using the 488 nm laser line and they all demonstrate excellent spectral separation.

Buccutite™ Rapid Antibody Labeling Kits makes preparing antibodies for flow cytometry quick and easy. Using a simple two step mixing protocol, researchers can directly conjugate PE, APC, PerCP or a tandem dye to any antibody or protein in less than 2 hours. No column purification is necessary, so you recovery is always 100%. Buccutite™ Rapid Antibody Labeling Kits are available in three sizes optimized for labeling 25 µg, 100 µg or 1 mg of antibody per labeling reaction.

Figure 3. Buccutite™ Rapid Antibody Labeling Kit workflow (figure drawn in BioRender).

Key features of the Buccutite™ Rapid Antibody Labeling Kits

• Can effectively and efficiently label antibody concentrations as low as 100 µg/mL (SMCC requires at least 1 mg/mL)
• Labeled antibodies are ready in under 2 hours, with as little as ∼15 minutes hands on-time
• Simple two step mixing protocol reduces batch-to-batch variation
• No column purification step required
• 100% conjugation yield
• Conjugates stable for long-term storage (4 °C, 12 months)
• Conjugtes ideal for multicolor flow cytometry applications

High-performance alternative to Lightning-Link™ Labeling Kits

Figure 4. Flow cytometry analysis of human PBMC cells. Anti-human CD4 antibody prepared with Buccutite™ Rapid PE Antibody Labeling Kit (Cat No. 1310) or Lightning-Link® PE. The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the FITC channel.

Table 2. Buccutite™ Peroxidase Antibody Labeling kits designed for labeling antibodies with HRP.

Additional Resources

Colorimetric, Fluorimetric and Chemiluminescent HRP Substrates

On it’s own HRP conjugates have little value, as they lack the capacity to produce a measurable signal. To facilitate target visualization HRP conjugates require compounds known as substrates. In the presence of hydrogen peroxide, HRP catalyzes the oxidation of these substrates to produce a quantifiable signal. HRP substrates are commonly divided into three categories according to they type of singal it produces, these include, colorimetric, fluorimetric or chemiluminescent substrates.

• Colorimetric substrates produce a visible color-change that can be measured using an absorbance microplate reader.
• Fluorimetric substrates produce a highly fluorescent byproducts that can be measured using any fluorescence instrument. Offers greater sensitivity then colorimetric substrates.
• Chemiluminescent substrates generate luminescent byproducts that can be measured using a luminometer. Since the emission of a photon is a result of a chemical reaction and not light excitation, there is minimal background interference.