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western blot problems - western blot troubleshooting (Apr/21/2009 )

I am a novice in this area and I am having a great deal of trouble with western blotting. I have tried WB with protein lysate with two different proteins and I get different problems everytime (i must have done this atleast 5-6 times). I use pre cast gels (Bio rad) and I don't have problems with the electrophoresis. During transfer, I use a dual color ladder and my PVDF membrane seems to have the colorful bands of the marker (my assumption is there was no problem with transfer). I use Supersignal Femto to detect my protein bands and I get poor results e.g multiple bands, faint bands of my protein of interest, bands that are not uniform/smooth (only part of the band is visible). I do a primary ab incubation overnight at 4 deg and block with milk + PBST (with 0.01% tween). I also get white blotches accross the membrane blocking some of my bands. While making the sandwich I remember to roll with a pipet to ensure that there are no bubbles. I am losing hope and my sanity. Please help!

-Distressed :wacko:

-pm1234-

pm1234 on Apr 22 2009, 12:21 AM said:

I am a novice in this area and I am having a great deal of trouble with western blotting. I have tried WB with protein lysate with two different proteins and I get different problems everytime (i must have done this atleast 5-6 times). I use pre cast gels (Bio rad) and I don't have problems with the electrophoresis. During transfer, I use a dual color ladder and my PVDF membrane seems to have the colorful bands of the marker (my assumption is there was no problem with transfer). I use Supersignal Femto to detect my protein bands and I get poor results e.g multiple bands, faint bands of my protein of interest, bands that are not uniform/smooth (only part of the band is visible). I do a primary ab incubation overnight at 4 deg and block with milk + PBST (with 0.01% tween). I also get white blotches accross the membrane blocking some of my bands. While making the sandwich I remember to roll with a pipet to ensure that there are no bubbles. I am losing hope and my sanity. Please help!

-Distressed :D


For faint bands, you could try exposing the film longer, but I suspect you may just have a bad Ab. Has anyone in your lab used this Ab for Westerns before? The white blotches are likely a result of pipeting the detection agent directly onto the surface of the membrane. Try pipeting it onto the edge of the membrane, then tipping it to disperse over the whole membrane.

-gfischer-

Make sure you don't over wash the antibodies, 3-4 washes of 5 min each in TBS-T is usually enough to remove unbound antibody. You may also need to try different blocking solutions - BSA, gelatine, maybe a bit of azide, can all help block. Have you titrated your antibodies to see what concentrations give you the best signal?
It seems kind of obvious, but how big is your protein? Are you transferring for long enough to get it onto your membrane?

From your description it does sound like you have a little problem with transfer, partial bands are usually the result of bubbles (maybe in the pads, not between the gel and membrane).

-bob1-

I don't really agree about the "over wash". I routinely wash for several hours at RT with incubation, and I get a perfectly fine signal each and every time. Its fair to say that I use pretty good Abs and that poor binding/poor specificity Abs would require shorter washes.

I would also guess that this is a case of "bad Ab". Is there any way you can try a positive control blot, such as actin?

-madrius1-