Odd Western Blotting problems - (Nov/21/2004 )

Hi all,

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.

Don Incognito

Try to drecrease the secundary antibody and wash conciously at least 5 times, 5 minutes each.

You as well can try:
- decrease loading of you protein samples (it helps often);
- include detergents in your primary and secondary Ab, like NP-40 up to 1%;
- decrease time of incubation with both Ab;
- wash membrane for all washing steps in strict washing buffer: 20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP-40; 0.1% SDS.

Good luck!

Alexei

I'm not sure why you air-dry your membranes before incubating with primary. I normally block the membrane in 2.5%-5% milk for 1h before I incubate in primary.
Also, I wash 3 times 20min both after incubating with primary and secondary...

Try that....

good luck

air-dry is good for dot plot, but I've never seen it in western