I generally use GAPDH, alpha-tubulin, beta-tubulin and vinculin as loading control for my western blotting experiments and gapdh for qPCR analysis. But recent publications show that in the hematopoietic cells, the cells of my interest, metabolism, cell migration are subjected to changes at various levels during their different stages of development or differentiation, where the above molecules especially GAPDH and vinculin are widely known to be implicated. In light of these facts, I am confused whether to use or not the above mentioned proteins as loading controls to normalize the gene expression across various genetic alteration conditions. It would be very great of you, if you can share your view and suggest some way out of this confusion.
Thanks.
Personal experience based on different gene manipulations (overexpression or knockdown) in mammalian cell culture, there is no single housekeeping gene.
For qPCR, better to make sure your input cDNA levels are the same and measure twice the RNA before reverse transcription. As housekeeping gene, you would give a try with Histone H3 and B2M also. whichever works best, you can go with that.
For Western Blot, you can also try Tubulin and Histone H3 in addition to yours. Also, I've seen that some papers have stained the membrane with Ponceau, took picture and put as control instead of a housekeeping protein. In this membrane, you would see the protein bands are pretty much similar. This was in a high journal as far I remember, either Science or Nature.
@ Dawkins-Hall, Zhang and Alptekin: Thank you very much for your suggestions. They are very helpful. Sorry for my late reply.
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