1. Prepare the assay buffer. The recommended buffer is 50 mM phosphate buffer (pH 6.0) containing 150 mM NaCl.
2. Dissolve avidin or streptavidin in this buffer at 0.5 mg/mL (final volume of 10 mL).
3. Prepare a biotin solution in assay buffer at a final concentration of 0.25 mM. Biotin is poorly soluble in aqueous solutions, so we typically dissolve the biotin powder first in a small volume of DMSO and then rapidly dilute it with buffer (pH 6.0) to the desired final volume. The solution should be stirred vigorously during this step.
4. Dissolve HABA in 10 mM NaOH at a concentration of 10 mM (2.42 mg/mL).
5. Prepare a biotin standard curve. Add 250 ^L of the HABA solution to 10 mL of the avidin or streptavidin solution. Incubate the mixture at room temperature for 10 min and then withdraw two 0.9-mL aliquots. Add 100 ^L of the phosphate buffer (pH 6.0) to each aliquot and determine the A500 of the samples. The average of these values represents the maximum A500 that the other samples will be compared to. Distribute 12-16 aliquots of 0.9 mL each of the HABA-avidin or streptavidin into reaction tubes. In duplicate, add increasing amounts of the biotin solution in a total volume of 100 ^L to the tubes to achieve final biotin concentrations ranging from 1.2 to 23 ^M (1.2 to 23 nmol of biotin per tube). The linear range of the assay extends beyond 23 nmol of biotin, but it is usually not necessary to include additional points. Stir the reactions for 5-10 min at room temperature and record the A500 of each sample. Average the duplicate readings at each biotin concentration and subtract these values from the average starting A500 value. Plot the differences on the ordinate versus the nmol of biotin on the abscissa. Unweighted linear regression should be used to construct the best straight line though the points to generate the biotin standard curve.
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