Antibody and Protein Labeling

Labeling an antibody with a specific tag has become an important and routine procedure in biological sciences and medical research. Tags, which range from visual markers such as fluorescent dyes, to hapten molecules like biotin, can be used to enhance the visibility of immune complexes and protein-protein interactions, or can be used in the isolation and purification of a protein.

When determining which type of label to choose focus on the downstream application for which the antibody will be used in. Fluorescent tags are used predominantly in cellular imaging, flow cytometry, fluorescent blotting and immunofluorescence applications, while enzymatic tags, such as HRP and AP, are commonly used for blotting, ELISAs and immunohistochemistry applications. Hapten tags such as biotin are the more versatile of the possible tag options as they can be used in protein isolation and purification, as well as, any of the aforementioned applications.

AAT Bioquest offers a comprehensive suite of antibody and protein labeling kits to choose from. Designed for the novice scientist in mind, these kits have the easiest protocols and require the least amount of hands-on experience.

Figure 1. ReadiLink™ antibody labeling workflow (figure drawn in BioRender).

ReadiLink™ Rapid Antibody Labeling Kits utilize amine-reactive labels to prepare covalently labeled conjugates. These reactive labels selectively target and bind to primary amines, such as lysine residues, on the antibody of interest. To label your antibody, simply mix your antibody solution (concentration ∼1 mg/mL) with the ReadiLink™ reaction buffer, add the mixture to the vial of pre-formulated label, and incubate for 30 to 60 minutes. Lastly, add the TQ™-Dyed Quench Buffer to your mixture and mix well. This part is key as it eliminates both background interference and the column purification step. After 10 minutes of incubation your conjugates are ready to use.

High-performance alternative to Lightning-Link® Antibody Labeling Kits

Figure 2. Flow cytometry analysis of human PBMC cells. Anti-human CD4 antibody prepared with ReadiLink™ Rapid iFluor488 Antibody Labeling Kit (Cat No. 1255) or Lightning-Link® Rapid Alexa Fluor ® 488. The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the FITC channel.

Table 1. ReadiLink™ Rapid Antibody Labeling Kit Selection Guide

BSA Compatible ReadiLink™ xtra Rapid Antibody Labeling Kits

ReadiLink™ xtra Rapid Antibody Labeling Kits with BSA compatibility takes labeling primary antibodies to a whole new level of convenience. Utilizing an optimized labeling protocol combined with vials of specially formulated labels, ReadiLink™ xtra kits enables rapid and reproducible labeling of microgram quantites of a primary antibody in the presence of common buffer additives and stabilizing proteins. In just two simple steps and without column purification, covalently label antibodies that are suitable for use in any immuno-based application.

Key features of the ReadiLink™ xtra Rapid Antibody Labeling Kits

• Compatible with BSA and other stabilizing proteins
• Label monoclonal antibodies, polyclonal antibodies or other proteins
• Labels 100 µg of antibody (two separate labeling reactions of 50 µg antibody)
• 100% antibody recovery, NO purification of the product is required
• Conjugates ready to use in 1 hr, with as little as ∼10 minutes hands-on time
• Choice of iFluor™ dyes, mFluor™ dyes, Alexa Fluor® equivalent AF dyes, FITC, Cy3, Cy5 or Cy7

Buccutite™ Protein to Protein Labeling Kits

Buccutite™ crosslinking technology offers a trouble-free way to covalently label proteins and antibodies with horseradish peroxidase (HRP), alkaline phosphatase (AP) or a phycobiliprotein. Compared to the conventional techniques for protein-protein conjugation, such as the SMCC chemical labeling method, Buccutite™ conjugation is significantly more robust and less demanding. These kits allow optimal labeling of microgram amounts of antibodies (as little as 25 µg per labeling reaction) in less than two hours, with no purification necessary and 100% yield. With Buccutite™ labeling kits, routinely prepare covalently-labeled conjugates that will withstand the rigorous washing techniques generally required in a typical immunoassay (e.g. ELISA, IHC and Western blot) or flow cytometry application.

Key features of the Buccutite™ Rapid Antibody Labeling Kit

• Label 25 µg up to 1 mg of antibody
• Choice of labels include HRP, AP, phycoerythrin (PE), allophycocyanin (APC), PerCP, PE-tandem dyes and APC tandem dyes
• Less than 2 hours of labeling time, with no purification required
• 100% antibody recovery

High-performance alternative to Lightning-Link® Antibody Labeling Kits

Figure 3. 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 3. Buccutite™ Rapid Antibody Labeling Kits

Product Ordering Information