B-actin as western blot -control - (Jul/11/2010 )
Hi! I am doing western blot to find the protein expression of my genes in normal and cancer tissues but I find that b-actin expression very less in normal and high in tumor samples. Usually since it is a house keeping gene, its expression level should be same in both normal and cancer tissue. Please help.
Although we tend to think the so called "house-keeping" genes should not change with conditions, they do. Perhaps b-actin does change in your tumor samples, if you are sure that you have loaded equal amount of protein...I would try it again, just in case i have done my maths wrong, or loaded more/less volume of the samples, or some how i haven't got an equal transfer...and if the problem persists, you might want to explore other house-keeping genes as loading control...
I think there unequal loading of proteins thats why beta actin not exact in each sample.
may be mistake in calculating protein conc . or in protein assay. check out this two possibilities..
WYF on Jul 13 2010, 04:00 PM said:
Yes equal amounts of protein was loaded, however where u require 7 microlitres for tumor, normal sample requires about 30 microlitres. does it effect when the wells are loaded almost full to its capacity? Will it affect the resolution and weaker bands?
ideally, you should adjust the volume of all your samples so that you load the same volume per well.