Bradford Vs beta-actin for WB protein quantification
Posted 25 June 2011 - 12:10 PM
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.
Posted 25 June 2011 - 05:41 PM
Posted 26 June 2011 - 12:56 AM
Posted 27 June 2011 - 07:05 AM