I am analyzing a protein with 50 kDa in platelet cytosol . I wanted to use b-actin as my loading control in Western analysis. However, b-actin and target protein migration distance were very close; therefore, I cannot reliable use b-actin as my sample loading control. Cytochrome c oxidase is variable in diseased platelets . What would be the next best loading control for platelet cytosol?
Thank you for your contribution to my question.
Regards,
baki
I recommend you consider total lane protein signal as your loading control. There are numerous reports showing it's as good or likely better than single protein normalization. I've used Poncea S after immunostaining data are safely collected, to get a total lane signal. Very easy and inexpensive. Coomassie also works and fluoresces in a NIR scanner-- a nice feature for LI-COR users. Bio-Rad stainfree gel UV generated signal also transfers over to the blot--though I find the UV inhibits transfer somewhat as it likely fixes the gel a bit, trapping the proteins in.
Hi Adam.
Thanks for the advise. If I use the total protein staining how do I choose the protein band for normalization. I am thinking that I need to look through all protein bands and pick one that does not seem to change. Am I correct?
Thank you
Baki
Hi Baki,
The idea instead is to use the whole lane (or much of it) as the normalizing signal, rather than relying on any one single protein. In this way, variations of a few dependent proteins are obscured by the overall stable expression of ten's or hundreds of others present in your lysate.
As a beginning point, search up Welinder and Ekblad from 2011, J Proteome Research. They show coomassie staining of blot membrane after immunostaining as compared to GAPDH. Though GAPDH tends to be the least reliable of the more commonly used normalizing proteins, it's still a valid point that normalizing against many will be more reliable than trusting one single protein to be stable under all conditions and treatments. There are many papers coming out on this topic. Have a look at related references to this paper as a beginning point of your investigation. I like this coomassie technique as it also normalizes for any irregularities in blot transfer, in addition to lane loading.
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