Preparation of Hydrazide-Activated Avidin or Streptavidin

The procedure for SPDP activation of phycobiliproteins can be found in Chapter 8, Section 1.7. Reacting the SPDP-activated phycobiliprotein with SH-labeled avidin at a molar ratio of 2 1 will result in highly fluorescent biotin-binding probes. 

The activation of avidin or streptavidin with adipic dihydrazide may be done using the method of Bayer et al. (1987a). A summary of this protocol is given below. 

In a test tube, dissolve 160 mg of adipic acid dihydrazide (Aldrich) in 5 ml of 0.1 M sodium phosphate, pH 6. Some heating of the tube under a hot-water tap may be required to help solubilize the compound. Cool to room temperature. 

Dissolve 50 mg of avidin or streptavidin (Pierce) in the adipic acid dihydrazide solution. 

Add 160 mg of the water-soluble carbodiimide EDC (Pierce) (Chapter 3, Section 1.1) to the solution, and mix to dissolve. 

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Hydrazide-activated avidin or streptavidin may be stored as a freeze-dried preparation without loss of activity. 

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