(All steps with agitation, unless done O/N at 4℃)

?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 4℃ 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 4℃ 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 4℃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.