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stripping too much?

western blot stripping

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#1 philman

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Posted 26 March 2012 - 01:15 AM

Hi,

I am doing some western blots, but seem to be having a little problem with getting my loading controls working. I have been looking at Phospho-ERK and phospho-p38, I am getting clear bands when I stain (albeit not much variation between the different samples, but that's a different problem) I am attempting to then strip my membrane to look at Total ERK or p38, or beta-actin, but then don't appear to get any bands.

I am using a strippping buffer I got off AbCam website:
20ml 10% SDS
12.5ml 0.5M Tris-HCl
67.5ml H2O
0.8ml beta-mercaptoethanol

then strip for 20 mins or so at 50 degrees C

After this I usually cannot see any bands upon staining with my second primary antibody. Or else I can see some faint bands that just appear to be in the same position and ratios as my first antibody, which I assume to mean it wasn't fully stripped.

Is my stripping buffer too strong? and if so, does anyone have any good suggestions for what is going wrong?

#2 knuf

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Posted 27 March 2012 - 08:48 AM

you could try a milder stripping. I think I got this also from the Abcam website, but my buffer is 0.2 M glycine, pH 2.5 + 0.05% Tween-20 warmed to 42C. I then incubate the membrane with the buffer with agitation for 10 minutes, then replace the buffer with fresh warmed buffer and incubate with agitation for another 10 mins. Then wash in 3x10 min in TBST, and incubate with the primary antibody. I have found that re-blocking the membrane is unnecessary usually. I've also found that this isn't very effective on really good antibodies, so I do the weaker antibody first.

Also, to test if your stripping was effective, re-incubate your membrane with the appropriate secondary antibody, wash, and develop. If you get a signal, then it wasn't stripped thoroughly and you should do it again. Sometimes its better just to run two identical blots or if you can, the cut the membrane horizontally and probe each protein separately--only do this if you have already used the antibody and know it doesn't cross react though.

Edited by kfunk106, 27 March 2012 - 08:52 AM.






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