For HIF1 alpha western blot, while doing the nuclear extraction, on following the volumes in the protocol (100ug tissue and 300uL solute) protein concentration in the samples prepared is too low. Can I increase the tissue weight to increase the sample concentration? Would that result in incomplete lysis?
For effective lysis you should have buffer/tissue ratio 20:1, so, you can increase amount of tissue at least 50 times.
Your tissue concentration is very low. So, you should augment the amount of protein. Also, I recomment you to check your antibody, which antibody concentration are you using? and if your antibody is the appropiated.
Thanks Prakash, Taras adn Osvaldo.
I did not get what you meant Taras, did you mean 20:1 or 1:20? If it is 20:1, buffer volume should be 20 times tissue volume which means to 100ug of tissue, i should be adding 2ml of buffer.
Osvaldo, I am using Novus anti rabbit HIF1a antibody concentration 1:1000
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