Bio-Rad Immun-Star™ AP Chemiluminescence Kits - Manual (Page 33)

1. Wash the membrane in 100 ml 1x BT solution for 10 min.
If the membrane has been in a buffer containing amines,
repeat the wash two more times.
2. Decant the wash solution and replace it with fresh 1x BT
solution. While agitating the incubation vessel, add 200 µl
of NHS-biotin. Incubate the membrane for 15 min with
constant agitation. Do not prepare the BT solution
containing NHS-biotin before use, as the biotin
reagent is hydrolyzed in aqueous solution.
Note: To prevent hygroscopic accumulation of water in
the NHS-biotin reagent, equilibrate the vial to room
temperature before use and remove reagent with a sterile
syringe.
3. Wash the membrane in 100 ml of 1x BT for 5 min. Repeat
the wash step.
4. Wash the membrane in 100 ml of TTBS for 5 min. Repeat
the wash step.
5. Prepare the avidin-AP or streptavidin-AP solution.
Incubate the membrane in the solution for 1 hr with
agitation.
6. Wash the membrane in 100 ml of TTBS for 5 min. Repeat
the wash step.
7. Wash the membrane in 100 ml of TBS for 5 min. Repeat
the wash step.
30