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GAPDH normalization
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: July 26, 2005 03:32PM
I keep reading journal articles that state the level of the protein of interest was normalized to GAPDH. i understand what this mean and that it is important to remove any loading error from the analysis. but how is it done?
Re: GAPDH normalization
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: July 26, 2005 07:15PM
I am not sure if you are asking how the normalization calculation is done. You can divid the densitometry reading of your protein by GAPDH reading. I think the membrane should be probed with antibody against protein of interest and then stripped and probed with GAPDH. This way you can control loading variations of your samples, assuming the stripping won't affect bound proteins on the membrane (PVDF). It is possible to combine both antibodies and perform only one western blot but I have never tried that.
Maybe I didn't get your question.
Re: GAPDH normalization
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: July 27, 2005 08:23AM
mitolab you did get the question. thanks for the answer. it was how i thought it was done.
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