Problems loading equal amounts of tissue lysate - This is driving me nuts!!!!! (Mar/07/2008 )
I'm trying to run out equal amounts of mouse tissue lysates (brain, heart, liver) to look at approx levels of my protein of interest in these tissues. Loading controls are causing me an absolute nightmare. Tubulin showing me loads more brain than the other tissues. F-actin looks equal and beta-actin similar to tubulin. I also have several x-reacting bands on the beta-actin blot despite seeing nothing of the sort on any of the blots on the Abcam website. Grrrrr.... I have quantitated with BCA twice and as far as I can tell, I've not calculated levels wrong. There are three of each tissue type and levels between these (ie all the brain samples) seem to be equal. Perhaps I extracted the tissues wrong, it was my first time and had to rush through it - used PBS + 0.5% triton x-100 .
Any ideas. Seriously thinking about giving up for a while!
You haver to show only one of these protein levels as control. all of them are not necessary. Many people use tubulin for brain samples. You could try to compare the levels of tubulin and load less amount of brain samples so that tubulin looks similar in all samples. Verify the brain sample concentration again.