Hello Everyone.
I see an enhanced expression/secretion of the housekeeping gene GAPDH gene in my transfected HEK293 free-style cells. I am 100% sure that I normalized the amount of loaded samples for the western blot but still, I see an increased amount of protein from day 0 to day 3 of expression. Does someone else also notice the same? Ideally, housekeeping gene expression should remain the same.
IDK why the mythos of a "housekeeping gene" still persists to this day that this gene set has to be stable in every tissue, every treatment, all the time. You should think of your traditional housekeeping genes, or reference genes as a shortlist of genes that are generally assumed to be stable but need to be validated in all your experimental conditions. Gapdh, Ppia, Beta-actin, Hprt, etc are decent sets to begin a reference gene study from which you will select a stably expressed a gene (or more than 1) for normalization purposes. I know from my own experience that Gapdh protein is not stable in the female hearts with aging, and I'm glad I didn't assume that to be the case. See "GAPDH—one of the most commonly used reference genes" in Article Reference genes in real-time PCR
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