Biotinylation of the Antibody

1. Determine the appropriate amount of photobiotin to use with Eqs. 1-3 in Subheading 3.1.1. Use a labeling reagent: antibody MR of 5.

2. Add the labeling reagent to the antibody and mix well. This should also be performed in subdued light.

3. Chill the reaction mixture on ice and irradiate it from above with visible light at a wavelength of 350 nm. The sample should be irradiated upright at a distance of 10-20 cm from the light source for 1520 min. The optimum wavelength of 350 nm can be obtained from a 250-W high-intensity mercury lamp. Sunlamps such as a Philips Ultrapnil (MLU300W), a National Self-Ballasted (BHRF 240-250V, 250W W-P), or a General Electric Sunlamp (RSM275W) can also be used. Appropriate eye and skin protection should be worn during the irradiation step.

4. Desalt the biotinylated antibody by dialysis or gel filtration.

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