Western blotting quantification and normalisation - (Dec/08/2003 )
I would like to quantify signals from western blotting.
1. Should I repeat a sample (positive control) between all my gels?
2. What's the right way to normalise my data? Should I normalise in relationship with beta-actin (house-keeping) and also with my positive control?
Thanks for all inputs
I have just finished a whole heap of westerns for my PhD. Beta actin was not an option for my work as its levels changed accros the samples i was using (so didn't make a good control). I ran coomasie gels of the protein loaded into each western adn used the densitometric value of the lanes in the coomasie as a stand for the densi results from the bands on my westerns. I also ran the western 3 times each to get a average densi value. Hpe this helps out.
Sorry but I personally do not like the Coomassie staining method as the linear range of the staining is very narrow. I use cdk2 for whole cell lysates instead of actin as sometimes actin levels change due to the shape and health of the cells.
Try cdk2 detection.... very strong and usually consistent.
and coomassie staining sensitivity is very low...0.5 ug
GAPDH could also be an internal standard
but I wonder know,
the steps in normalizing my bands across 2 comparing samples using the internal standard...
since protein loading doesn't perfectly the same in each time...so the band of internal control could vary slightly
thanks very much for any opinion