My target proteins are RyR2 and SERCA2 and their respective regulators.
If you are not interested in gender or sex differences, actin and tubulin both should give you a reliable normalization.
Julius, I recommend not using housekeeping proteins for normalizing western blots. There is much literature to show this is likely to skew your data. Read up on "total protein" as a loading control. Pre or post stain your blot (not gel) to visualize the whole lane protein and use that signal as your control. I'm using Ponceau as a quick easy way to visualize the blot. It can be done before or after immuno staining. There are also reports of Coomassie being used. No housekeeping protein is reliable without careful empirical testing in your specific conditions.
Thank you Professor Choubey and Mr. Munhall for your answers.
Mr Munhall, I hope it isn't too much but would you be able to share a (your?) protocol on this method? I have never encountered such data analysis in my lab but I have seen papers on SR vesicle immunoblotting that presented their data the same way as you have mentioned. Would a luminescence image analyzer (LAS 3000) be enough for this procedure? Thanks!!!
I nice reference that discusses a couple different methods for total protein normalization is included in Gilda and Gomes study, Methods Mol Biol. 2015;1295:381-91. doi: 10.1007/978-1-4939-2550-6_27. I have used the Bio-Rad version of so-called stain-free gels to explore this method, but found wet transfer from a UV exposed gel was someone less efficient compared to non-UV exposed gels. Presently, I'm using Ponceau S for 20 minutes on the blot after immunostaining to visualized, then scanning the blot on a basic flatbed scanner (with gamma adjustment turned off ie. set to 1.0). These methods work easily enough if 1) your total blot lanes are clear of any irregularities (bubbles etc) and that the lysates run on each lane are from a similar source. I was not able to use this method when running lysates from various labs of various species even though I was given their BCA protein concentration assay values. The band patterns are very different in this case and don't seem to sum up equally--though theoretically they should.
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