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western blot loading control


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#1 fabipp

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Posted 10 February 2012 - 06:04 AM

When I am using loading control antibodies in my western blot (I am suing CDC-42 and GAPDH) I see multiple bands. CDC 42 has the MW of 21 but I see a very strong band at 43 kDA and more some not soo strong inespecific bands. GAPDH I also see inespecific bands. Can be because I am not using a protease inhibitor when I am extracting proteins. I am extracting proteins from Candida albicans. It is diffcult to fing a loading control. I found tubulin, the signal was perfect, no unespecific bands, but I am working with Heat Shock Proteins, so Tubulin is also activate tubulin, so I cannot use tubulin to control teh loading of my proteins. with beat actin I got a very weak signal and a very high background. Wating for an answer, please.

#2 bob1

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Posted 11 February 2012 - 10:25 AM

Protein degradation usually (actually always) results in bands that are smaller than your target protein or a smear, or nothing in your lane. I would always use protease inhibitors for making lysates as some of the proteases are very fast acting.

It sounds more like you are having specificity issues with the antibodies, which are probably not designed for yeast work - try titrating the antibodies out to minimise non-specific binding. You could also work try more stringent conditions - higher salt and/or more detergents in the antibody incubation and wash steps.

#3 mdfenko

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Posted 14 February 2012 - 12:36 PM

you may also be looking at polymers of the proteins (43 is probably a dimer of 21).

you may also be looking at non-specific staining of a common artifact which appears in the 50s-60s range (may also be in the 40s).
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