Bradford Vs beta-actin for WB protein quantification - (Jun/25/2011 )
I have discrepancies between how much protein should be on my nitrocellulose western blot membranes according to Bradford quantification and what beta-actin tells me. Briefly, here is what's happened:
1 - Tumour tissue protein extracts were quantified using Roti Nanoquant (modified Bradford technique - traditional Bradford gives almost identical results).
2 - Sample volumes were adjusted in order to load 40 micrograms of protein onto 10% SDS-PAGE gels.
3 - Gels were transferred to nitrocellulose membranes using a wet transfer method (110V for 60 min)
4 - Membranes were probed and stripped twice (using a tested protocol) before being produced for beta-actin.
According to the beta-actin western blot, the protein loading is quite variable. However I am reluctant to normalise my western blots to actin given that I've quantified the proteins with Bradford. I stained the membranes with Ponceau Red post transfer and all lanes show equivalent loading, I've also stained the post-transfer gels with Coomassie R250 stain that from the quick look I had, the residual protein (mostly high molecular) seems consistent (although I will double check this). Given that I've got three lines of evidence suggesting that the protein loading is equivalent and beta actin tells me different, I am unsure of which to trust more. I do know that there are doubts about whether beta actin is an appropriate loading control (especially when dealing with samples from multiple patients).
Any thoughts of what I should be trusting, or anything I can do to be sure of the protein loading?
Cheers in advance.
I don't trust stripped results - how do you know that the stripping occurred evenly over the whole blot? In my experience stripping is quite variable, and if performed more than once, damages the protein on the membrane so that you get uneven results.
Indeed, stripping shouldn't be used to quantify different proteins since some proteins may be stripped faster or slower than others. I'm interested in relative differences of the same protein across patients. Under the same conditions the same protein should be stripped at the same rate, and to be sure I've probed a membrane twice with the same antibody after multiple strippings and got the same result.
I would definitely be skeptical of the actin as a proper loading control as cytoskeleton is definitely dynamic and can and will change in response to some conditions. Try a different housekeeping protein and if it gives you the same variable results, it is probably right.