A New Protein Crosslinking Method for Labeling and Modifying Antibodies

Introduction

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The covalent conjugation of a large biomolecule to another macromolecule, such as an antibody to an enzyme, or an enzyme to DNA is an important task for researchers. There are a few methods available for crosslinking two biomolecules. One common method is to use a small crosslinker (such as Sulfo-SMCC). SMCC has an amine-reactive NHS ester group at one end to react with free amines (-NH2 ) of a protein, and a sulfhydryl-reactive maleimide group at the other end to react with a thiol group (-SH) of biomolecule to be conjugated. SMCC-modified linker on a protein is extremely unstable and often self-reactive since proteins often contain both free amines and thiol groups, resulting in a significant amount of homo-crosslinking. In addition, the quantitation of the number of maleimide groups on a protein is quite tedious, and it is difficult to set the suitable molar ratio of two biomolecules to be linked in the reaction for the desirable function and coupling efficiency.

A convenient and effective crosslinking method has been developed to link two biomolecules with a high conjugation yield. This method uses two unique crosslinkers (Buccutite™ MTA and Buccutite™ FOL). The crosslinkers of MTA and FOL on proteins are easy to control and quantify. Following a desalting column to remove free linkers, and the Buccutite™MTA activated antibody and Buccutite™ FOL modified protein readily react upon mixing under a mild condition. The purification is essentially unnecessary, and there is no homo-crosslinking of each biomolecule in the reaction.

 

Materials and Methods

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• Antibodies and Enzymes: Goat-Anti-Mouse IgG and mouse IgG are from Jackson Immuno Research Laboratories; HRP is from Calzyme, Tubulin mouse mAb is from Life Technologies.
• Purification Columns: All purification columns are Bio-Spin size exclusion columns from Bio-Rad.
• Cells Imaging: HeLa cells are fixed with 4% formaldehyde, and stained with Mouse Anti-tubulin, and then stained with the HRP-Goat–Anti-Mouse IgG conjugate. Images were taken with Olympus IX71 fluorescence microscope.

Conjugation Principles

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1. Goat-Anti-Mouse IgG was activated with MTA , and tag protein (APC or PE) was modified with FOL (1hour).
2. The activated IgG and modified tag protein was purified with a spin column (5min).
3. Mix the activated antibody and modified tag protein (30 min).
4. Stain cells with the desired conjugate without further purification.

Results

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Buccutite™ HRP-IgG Conjugate for ELISA Assays

Buccutite™ IgG-RPE Conjugate for cell imaging

Summary

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• Linkers are highly stable. The Buccutite™ linker-activated macromolecules are very stable, can be stored at 4ºC for more than 24 months.
• Conjugation conditions are extremely mild and robust. Buccutite™ conjugations can be run in a broad range of pH, concentration and temperature. No catalyst is required.
• High yield: Buccutite™ conjugation can be finished within 1 hour. Buccutite™ conjugation gives much higher yield than other existing methods under the same conditions. The conjugation can be run at extremely low concentration.