HRP Antibody Labeling Using Buccutite™ Crosslinking Technology

Protein-protein conjugations are commonly performed with a bifunctional linker (such as the commonly used SMCC), having different reactivity on each end for linking two different proteins. One end of the crosslinker reacts (via NHS ester) with amines (-NH2) found in the amino acid lysine and N-terminus, and the other end reacts (via maleimide) with the thiol groups (-SH) found in the amino acid cysteine. However, SMCC-modified protein is extremely unstable and often self-reactive since proteins often contain both amine and thiol groups that cause significant amount of homo-crosslinking. In addition, it is quite difficult and tedious to quantify the number of maleimide groups on a protein.

Buccutite™ crosslinking technology provides a new robust method to label proteins with another macromolecule (such as proteins, nucleic acids, oligos, peptides or other biopolymers). Buccutite™ crosslinking technology uses two small linking molecules that readily react with proteins through their amino groups (N-terminal or lysine residue), and the two resulted Buccutite™ pairs of macromolecules can be readily cross-linked by a simple mixing as shown in Figure 1.

Based on this new Buccutite™ crosslinking technology, ReadiLink™ Peroxidase (HRP) Antibody Conjugation Kit (Cat# 5503) has proven to be one of the most robust HRP labeling kits. The kit is designed for preparing horseradish peroxidase (HRP) conjugates directly from proteins, peptides, and other ligands that contain a free amino group. The HRP provided has been pre-activated with our proprietary linker Buccutite™ FOL, and can be directly used for conjugation. The Buccutite™ FOL -activated HRP readily reacts with Buccutite™ MTA-containing molecules under extremely mild neutral conditions without any catalyst required. Compared to commonly used SMCC and other similar technologies, our Buccutite™ bioconjugation system is much more robust and easier to use. It enables faster and effective conjugation of biomolecules with higher efficiencies and yields.