Rapid Protein Crosslinking Method for Labeling and Modifying Antibodies

Crosslinking is a technique that chemically joins two or more proteins together, such as an antibody and an enzyme, by a covalent bond. Crosslinking reagents consist of reactive moieties that modify and attach to specific functional groups on proteins. A common approach for conjugating two biomolecules utilizes a small, heterobifunctional crosslinker called succinimidyl 4-(N-maleimidomethyl) cyclohexane1-carboxylate (SMCC). SMCC crosslinkers contain amine-reactive NHS ester moieties and thiol-reactive maleimide moieties to crosslink proteins. However, a drawback to this approach is the many experimental conditions that must be monitored to perform a successful conjugation. Difficulties associated with SMCC conjugations are maintenance of a narrow pH range, self-polymerization, and hydrolytic degradation of reactive moieties. To address these concerns, AAT Bioquest has developed an optimized and robust Buccutite™ crosslinking technique. Buccutite™ crosslinking technology provides a simplistic and efficient approach for conjugating proteins. It can be completed in half the time and with less stringent parameters yielding highly stable and easy to handle conjugated proteins.

Buccutite™ Crosslinking Principles

Conjugate Proteins In 2 Simple Mixing Steps. No catalyst required

Buccutite™ vs SMCC

Total Operation Time:

• Buccutite™: 2 Hours
• SMCC Chemistry: 4-8 Hours

Minimum Sample Concentration

• Buccutite™ Chemistry: ≥0.5 µg/µL
• SMCC Chemistry: 0.5 - 5.0 µg/µL

Optimal Conjugation pH:

• Buccutite™ Chemistry: 5 - 9
• SMCC Chemistry: 7.0 - 7.5

Conjugation Yield

• Buccutite™ Chemistry: 50-60 %
• SMCC Chemistry: ≤ 30 %

Advantages

Buccutite™ conjugation can be run in a broad pH range of 5-9 and temperature.

Fast: Buccutite™ conjugation can be completed in 1-2 hours under extremely mild conjugation condutions. No purification step required with 25μg kits.

Robust: Buccutite™ conjugation can be run in a broad pH range of 5-9 and temperature.

Highly Stable: Buccutite™ linker-activated macromolecules are very stable, can be stored at 4ºC for more than 24 months. 

High Yield: Buccutite™ conjugation gives much higher yield than other existing methods under the same conditions. Eliminating homo-crosslinking of proteins.

Tubulins were imaged with RPE- goat-anti-mouse IgG conjugate in HeLa cells. Tubulins were stained using mouse anti-α-tubulin antibody, and visualized with red fluorescent RPE-goat-antimouse IgG conjugate prepared by Buccutite™ crosslinking technology as described above.

AAT Bioquest’s Custom Bioconjugation Services

AAT Bioquest offers same-day custom conjugation of proteins or antibodies with a wide array of labels, such as biotin, HRP and over 20 fluorophores. Our services are designed to produce high-quality results with a fast turnaround. We take pride in supplying scientists with flexible tools that help expand their research needs.

Features of AAT Bioquest’s custom bioconjugation services include:

• Guaranteed quality with 95% purity rating
• Affordable price tiers that work with your budget
• Scalable service with minimum order of 50 µg
• Same day order fulfilment means your conjugate ships the day you order it

*NOTE: APC and PE custom conjugation services require 3 working days You may supply your own protein or antibody or chose from 3000+ monoclonal and polyclonal antibodies in our catalog. Contact us for a quote today, and let us put our expertise to work for you!

Product Ordering Information For Buccutite™ Labelling Kits