Tips for Western Blot of Low Molecular Weight Protein (HCV core protein) - (Oct/04/2009 )
I am working on a project in which I am doing Western Blots to look at the expression of HCV core protein with HA-tag (18 kDa) in Huh7 transfected with a plasmid expressing HCV core. I have been using a 0.2 uM PVDF membrane for my transfers and I have been transferring for 90 minutes at 70 mA in semi-dry system. I have been getting nice Ponceau S staining, so my transer appears to be working. In addition, the prestain protein marker is transferred to PVDF well. I use anti-HA antibody (12CA5) as primary antibody in western blotting. However, I have not been able to detect HCV core protein with HA-tag. I was wondering if anyone had any tips regarding doing Westerns of low-molecular weight proteins. I've been using a 15% gel for SDS-PAGE. I would appreciate anyones helps. Thanks.
I've done western blots to probe for mono ubiquitin (~8.5 kDa) using nitrocellulose membranes. Because the protein is so small, our lab boils the membrane (~15 minutes in the microwave) to denature the low mw proteins as much as possible to allow for more binding by the antibody. I'm not sure if you can boil PVDF membranes, but nitrocellulose ones are more fragile and if they can boiled then I'd guess PVDF ones can be too.
Hope that helps