I've been running westerns for months with few problems.
Very recently, my blots have started to show multiple nonspecific bands and a great deal of background, even with guaranteed high-quality primaries (such as Sigma's mouse anti beta-Actin, which normally gives me no background whatsoever).
My protocol is (has been) as follows:
-- Proteins are transferred from 12% polyacrylamide gels to PVDF membranes via semi-dry or wet transfers.
-- Blots are air-dried before incubation with primary antibody (in 5% skim milk and Tris-buffered saline containing 0.05% Tween-20) at room temperature for 1 hour.
-- 3 washes, 10 minutes each.
-- Incubation with secondary at 1:5,000 - 1:10,000, with or without skim milk (depending on the primary) for 1 hour at R.T.
-- 2 washes, 5 minutes each, before adding ECL or ECL+, etc.
So far, I've verified that the problem isn't with the primaries (they are various, and functional) or secondaries (as control blots without primary do not give background problems). I've made new buffers and solutions, but to no avail. Protein degradation has been suggested as a possible reason, but I don't get the smear of protein with molecular weight equal to or less than that of my target proteins (which are rather small, anyway).
Any help would be appreciated.
Edited by DonIncognito, 22 November 2004 - 05:49 AM.