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Avidin - Assay

Source:
Egg White
CAS:
1405-69-2

Avidin Assay

Method
The biotin-binding capacity of avidin is determined using the spectrophotometric method of Green (1963). One unit will bind one microgram of d-biotin at pH 8.9.
Reagents
  • 0.2 M Ammonium carbonate, pH 8.9
  • 0.001 M d-biotin (24.4 mg/100 ml) in ammonium carbonate buffer, pH 6.9
Enzyme

Dissolve at 0.2 mg/ml in 0.2 M ammonium carbonate buffer, pH 8.9.

Procedure

Adjust spectrophotometer to 233 nm.

Add 3 ml of stock avidin to control and test cuvette. Add 10 ul of d-biotin to test cuvette and record ΔA233. Continue adding 10 ul biotin at a time to the test cuvette until no further increase in A233 occurs after three successive biotin additions. Plot the number of aliquots of biotin used versus A233. Determine the equivalence point by drawing a straight line through the linear absorbance portion of the curve and by drawing a line through the portion where no further increase in A233 is observed. The intersection of these lines is the equivalence point. Determine the number of aliquots represented by the equivalence point.

Calculation