(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.