B- actin is used a great deal for quantitation of liver lysates. What other alternatives are there for WB analysis? should we opt for GAPDH or tubulin?
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1$\begingroup$ This is totally up to you, and the antibodies you have on hand. The quality of the antibodies helps as well. There are many so-called "housekeeping" genes whose expression is not necessarily greatly affected by various conditions. Beta-actin, alpha- and beta-tubulin, GAPDH, COX IV, Histone H3... all work well, and some are better in some scenarios than others. For example, H3 would likely be a much better loading control for nuclear lysates than actin or tubulin (even though both do occur in the nucleus in some circumstances), due to its much higher and more stable expression. $\endgroup$– MattDMoMar 19 at 22:01
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$\begingroup$ So, test what you have, and ensure that it truly isn't affected by your treatment or disease conditions. Also, make sure that the signal isn't saturated, otherwise you can't quantitate results and draw meaningful conclusions about your data. $\endgroup$– MattDMoMar 19 at 22:02
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$\begingroup$ I'm not posting an answer as I've voted to close this question as opinion-based. This is not a discussion forum; rather it is a Q&A site for biology researchers, academics, and students where specific objective questions relating to biology get specific objective answers. Please take the tour and carefully read through the help center to learn more about the site, including what is on-topic and what is not, and how to ask a good question. $\endgroup$– MattDMoMar 19 at 22:05
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$\begingroup$ @MattDMo, I think there's an objective answer to the question "What other alternatives are there for WB analysis? ". $\endgroup$– Michael_AMar 20 at 2:13
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Use a total protein stain. Irrespective of tissue type, normalising your immunoblot signal using a total protein stain is a good, probably superior, approach (ref1, ref2, ref3). Many journal guidelines now strongly encourage the practice;
Normalize signal intensity to total protein loading (assessed by staining membranes for total protein) whenever possible. “House-keeping” proteins should not be used for normalization without evidence that the experimental manipulations do not affect expression.
If you really need to use single protein loading control, confirm that it's stable in your hands; the abundance of any given protein in a tissue could still be perturbed by your experimental conditions.
Confirming consistent loading of your lanes using a total protein stain can help to confirm that your loading control is consistent in your experiment. I generally collect both total protein signal and a loading control but will only use the latter if a reviewer is being difficult.
