Different beta-actin levels - (May/26/2009 )
I was trying to see SR-BI expression level from different cell lines. I performed western blot and loaded the same amount of protein (1 ug). The result was what I expected: cell lines that were supposed to express SR-BI had bands and others not. The problem is that beta-actin level fluctuates greatly among cell lines. I thought it was because I loaded such small amount of protein that a small pipetting problem can change the protein level, but when I repeated the western blot, they come out in the same pattern (same cell lines have higher beta-actin level). I can use imaging programs to normalize the data, but I'd like a better picture. What other proteins can I use for normalization and is there any documented report on the different beta-actin levels in different cell lines?
sihyunie on May 26 2009, 03:18 AM said:
It makes sense that different cell lines would have different expression levels, based on a number of factors. You could also try GAPDH.
It is a well known phenomenon - different tissues and hence different cell types have different levels of actin; gapdh, isn't much better. Try loading comparable cell numbers - i.e. count your cells and lyse on a cells per ul basis.
you could probably just load the same amount of protein (as per bradford or BCA) in each lane.
this is similar to counting cells but allows you to do it after you lysed your samples (assuming that you didn't lyse them in loading buffer straight away).