Biotin – N-Hydroxysuccinimidobiotin (NHS – Biotin) # 20217 (Pierce). Or
NHS – LC –Biotin), # 21336 (Pierce)
- Dialyse antibody in 100 mM NaHCO3, pH 8.3. Dialysis can be performed more quickly if 100 volume changes are used –3 X 30 mins
- Adjust the concentration of your antibody to 2mg/ mL
- Make up biotin solution 10mg/mL of DMSO immediately before use. You don’t need 1 mL of biotin solution, so weigh a few mg of biotin and make it up to 10 mg/mL (e.g. 3.4 mg Biotin add 340 m L DMSO).
- Try a range of 10 –400ug biotin per mg of antibody (10, 40, 80, 160ug /mg).
- Add biotin to antibody, mix well.
- Wrap the tube (s) in foil, incubate and rotate at room temperature for 4 hrs.
- Dialyse antibody in PBS. I used Centricon column, adding 1 mL PBS, spinning at 5000 g for 15 mins and repeat 3 x.
- Compare each conjugate by staining (perform a titration of antibody on cell for each conjugate (1/5, 1/50, 1/100, 1/200, 1/400…) to determine the optimal biotin and staining concentration.
- Add BSA to 1% and preservative sodium azide (NaN3) to 0.02%-0.04% and store at 4 ° C