Loading Control Calculations

[KKM]

I am using Actin as a loading control for my WB. I am running rat brain. I do see slight changes in Actin that I'm confident are not related to our experimental conditions. I expect that they are due errors in protein extraction, concentration analysis and/or loading that are much more prevalent in tissue than in cells, thus making normalizing against loading controls necessary. I want to know how to account for the changes in Actin in my calculations. I have a positive control in all my membranes. Do I make all the wells equal to the positive control for Actin levels and then subtract that factor from my protein of interest density levels, essentially treating it as background? Eg. if my control well Actin density is 20.9, and an experimental well Actin density is 40.8, do i just subtract 19.9 from my experimental well protein of interest density?
Thank you!

[Amy Coll]

Hi KKM,
Q: are you normalising the protein concentration prior to loading, using a BCA protein assay for eg? Then you will load exactly the same concentration is each well, and therefore the same band intensity should be seen.
I presume that you are restriping the membrane for actin expression, and I usually minus the background signal and divide by actin to give a ratio of test protein expression.
I have also seen that stimulations with some cytokines, such as TNF can alter actin expression and an alternative endogenous control was used.