question about protein expression normalization. - (Sep/26/2012 )
Hi everyone, I am a new member and I found this site is really helpful.
When I normalize (intact vessels) my samples, should I choose actin or the total protein?
I was told that I should use total protein in the case if actin was affected by any treatment or condition, am I right?
I can certainly see why total protein would be useful since sometimes a protein assay still gives me crappy B-actin results.
I imagine total protein probably has its own limitations though. Can't a treatment change total protein just as it changes actin?
You could choose either, however there are also other genes that are commonly used for normalization such as tubulin and nucleolin.
Hi Thelomitra pulchella
Yes we can use either. However, some people says that the total protein is more accurate than the actin, tubulin.
Total protein is only as accurate as the assay you use to measure it. Having done westerns for quite a few years now, I would say that the accuracy of most protein content assays is somewhat questionable due to the chemistry of the assay. For instance in the BCA assay (one of the more reliable) the values obtained for different proteins depend on the number of cys-cys, tyr and trp residues in the protein... if you use BSA as a standard, your total protein values are not accurate (though they might be precise)! Then you have to ensure that your samples lie within the reference range and that you are not exceeding the capabilities of the sectrophotometer used to read the assay, as well as ensuring that you have used the appropriate standard curve.
Quantitation itself is problematic as it depends heavily on correct exposure of the film, and a number of variables during the blotting process such as even transfer at all MW, affinity of the antibodies for the different proteins.
You might be better off normalizing to bands from a ponceau stain of the membrane.
if you're procedure leads to crappy actin, tubulin, gapdh, or any of the other common loading controls then that's reason to suspect your results at large, hence normalizing to total protein not exactly helping you out there unless you just ignore the bad house keeping control results
you can get a "sampler" pack of the various house keeping proteins and probe/strip/reprobe to see which one works best for you...