Unfortunately, a standard protocol is not available for this product. The sample binding capacity protocol below may be helpful:
1. Pipette 2.0 mls of resin into pre-calibrated, fritted columns and allow the resin to pack. Measure the packed volume in milliliters.
2. Wash the resin with 5 packed volumes of 0.5 M sodium chloride.
3. Equilibrate the resin with 25 ml packed volumes of phosphate buffered saline (PBS), product P4417. Check the pH of effluent after the wash. The pH should be 6.75 to 7.25. If necessary, equilibrate with additional 25 X packed volume.
4. Dissolve avidin from egg white, product A9275 at 80 mg protein/ml in PBS. Check the pH and record. If necessary, adjust the pH to 6.95 to 7.05 with 0.1 N HCl.
5. Determination of Avidin load:
PBS 10.0 ml
Avidin at 80 mg/ml 0.05 ml
Mix by inversion and measure A (280 nm) versus PBS.
6. Calculation of Avidin Load:
[(Net Absorbance (280 nm) x 10. 05]/(0.05 x 1.54)
7. For each ml of packed gel in the column, add 1.0 ml of the avidin solution. Allow the column gel to equilibrate in the presence of the avidin for one hour with intermittent swirling.
8. Wash the column with 100 packed volumes of PBS. Mix each wash by inversion and measure A (280nm) versus PBS.
Calculation of wash:
[(Net Absorbance (280nm) x 100] / (1.54)
9. Bound avidin to biotin agarose= mg of avidin labeled - total avidin in wash (unbound).
10. Binding capacity = (Bound avidin to biotin-agarose)/ml of packed gel.
Additional references and general protocols are also available at the links provided below:
https://www.sigmaaldrich.com/search/b0519?focus=papers&page=1&perpage=30&sort=relevance&term=B0519&type=citation_search
https://www.sigmaaldrich.com/applications/protein-biology/protein-pulldown