Protocol: Minimal Biotinylation of Proteins

This protocol is used to minimally biotinylate proteins for capture on a strepavidin or neutravidin sensor chip. Capture of biotinylated proteins provides a more uniform orientation of proteins on the surface than does amine coupling.

You will need:

• EZ-Link® Sulfo-NHS-LC-LC-Biotin, Pierce, cat no 21338 (Manufacturer's information)
• Three 50 µl aliquots of protein to be biotinylated at a concentration between 1 - 10 mg/ml. The protein must be in a non-amine containing buffer.
• Ice water bath
• Zeba Desalting Spin Column, Pierce, cat no 89882 (Manufacturer's information)

Procedure:

• Biotinylation is a random event. In order to protect the binding site of some proteins, it may be necessary to biotinylate in the presence of the binding partner and then dissociate.
• Weight out approximately 1 mg of EZ-Link® Sulfo-NHS-LC-LC-Biotin and add appropriate volume of water for a 1 mg/ml solution. Use immediately.
• To one aliquot of protein, add 10 µl of EZ-Link® Sulfo-NHS-LC-LC-Biotin (A). To the second aliquot, add 2 µl of EZ-Link® Sulfo-NHS-LC-LC-Biotin (B), and to the third aliquot, add 0.4 µl of EZ-Link® Sulfo-NHS-LC-LC-Biotin (C).
• Incubate in ice for 30 min. Other incubation times (30 - 120 min) or temperatures (room temp) may be used if there is difficulty in biotinylating the protein.
• While the proteins are incubating, prepare the Zeba desalting columns. The columns contain sodium azide and must be rinsed thoroughly. Add 200 µl of water or buffer and spin in a µfuge for 2 minutes at 2000 rpm. Repeat four (4) more times discarding the eluate. Keep the column clean and moist until the protein is added.
• After incubation, carefully pipet the protein samples into three Zeba desalting columns. Spin in a µfuge according to the instructions that accompany the Zeba desalting columns. This step is necessary to remove free biotin from the sample.
• Recover the protein and store appropriately.
• Not knowing the extent of biotinylation, start with capture of tube (C) to strepavidin (neutravidin) surface. If the desired capture is not achieved, continue with tube (B). If still not high enough, finish with tube (A).