Assessing differential expression of activation-related proteins in naive versus activated T cells is hampered by the fact that apples are compared with melons. Activated T cells vastly increase cell size, cytoskeleton, ER, protein synthesis and metabolism.
A problem occurs when you try to normalize to an internal control like actin, tubulin, Gapdh, etc. Despite equal loading according to Lowry determination all these proteins show increased expression in activated T cells a result understandable in light of the huge changes these cells undergo. What causes the bias? What would be a good loading control in this particular case? Histones, HDAC1? Can someone help with suggestions?
Dear Bernhard, this is really interesting problem. In our experiments, beta-2-microglobulin was more adequate control than GAPDH in Real-Time PCR. I am not confident that this will be true for Western blot, but beta-2-m deserves attention. Good luck!
Dear Dmitry, Thank you uvery much for your helpful comment. We tried beta-2M but still a quite striking discrepancy remained. If I find the solution I will post it.
I am having the same problem at the moment. I have done a real time study and did a genorm analysis on 8 normalisation genes and ended up running with two for the study but TATA binding protein (TBP) looked the best between Naive and activated samples. I haven't tried it for protein loading yet as I may not need it. Be interested to see how you get on for future reference.
Joseph, Thank you very much for the TBP suggestion. I used TBP in qrtPCR normalisation of beta cell messages and have the primers for it. Your are right, a TBP antibody might do the trick. I keep you posted, Bernhard
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