Chen-Ming Fan Lab,Carnegie Institute of Washington

(All steps with agitation, unless done O/N at 4oC)
Rinse membrane once in TBS-T
Block the membrane with TBS-T/ 5% milk (or HIHS) for 30 min room
temp (can be left at 4oC O/N here)
Rinse membrane briefly 2X with TBS-T and then once for 10 min with
TBS-T
Add primary antibody diluted into blocking solution. Incubate at room
temp for 1 hour. (Can be left at 4oC O/N here)
Wash briefly 2X then 3X10 min each with TBS-T
Add secondary HRP conjugated antibody diluted into blocking
solution. Incubate at room temp for 1 hour. (Can be left at 4oC O/N
here)
Wash briefly 2X then 3X10 min each with TBS-T
Mix an equal volume of detection reagent 1 with detection reagent 2
(usually 2mLs total is sufficient).
Drain excess TBS-T from membrane. Lay membrane on a piece of
parafilm. Pour on detection reagent mixture. Lay a second piece of
parafilm over the top being careful to avoid any bubbles.
Incubate for 1 minute (no agitation)
Drain off excess reagent and wrap membrane in saran wrap. Do not let
the membrane dry!
Quickly, expose membrane to film. Test exposure times of 30 sec, 2
min, 10 min etc.