Activation & Capture
This protocol is used to activate a pre-conditioned CM sensor chip for amine coupling. The sensor chip can be used as is for amine coupling of ligands or strepavidin or neutravidin can be coupled to make a SA (NA) chip for capture of biotinylated compounds.
You will need:
- a fresh CM chip which has been pre-conditioned
- N-hydroxysuccinimide (NHS) which has been diluted and aliquoted. Individual vials are found in the freezer
- 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) which has been diluted and aliquoted. Individual vials are found in the freezer.
- An empty vial
- Ligand or strepavidin/neutravidin. DO NOT PREPARE IN ACETATE BUFFER UNTIL IMMEDIATELY BEFORE USE!
- For strepavidin, prepare immediately before use, 2 mg/ml (1 mg/500 µl acetate buffer) solution of strepavidin in acetate buffer, pH 4.5. Centrifuge in table top ultracentrifuge at 100,000 rpm, 4°C, for 10 min. Avoiding any pellet, transfer immediately to a 7 mm vial and cap.
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:
- 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
- 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.
- Weigh 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. The goal is minimal biotinylation.
- 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 1,500 x g (4800 rpm in lab microfuge). 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. Notice that the manufacturer recommends adding 15 µl of water or buffer to column before adding sample if the volume is less than 70 µl. 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).
Protein calculation using the Waddell method*
This methods assumes that you have an accurate spectrophotometer and that the buffer does not absorb in the far UV range.
Blank the spectrophotometer from 200 nm to 240 nm (if you have a scanning spectrophotometer) with a suitable blank and read the blank afterwards. Measure the absorbance of the peptide solution at 215 nm and 225 nm. The absorbance at 215 nm must be less than 0.5; if not, dilute with buffer and measure again. Measure the absorbance at both 215 nm and 225 nm. To determine the mg/ml: A215 – A225 * df * 0.144.
Divide the mg/ml by the MW of the peptide to determine the molar concentration and multiply this result by 1,000,000 to determine the µM concentration.
*W.J. Waddell J. Lab. Clin. Med, 48 (1956), pp. 311-314.
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