Westerns with serum isolations - What's your protocol? - (May/07/2008 )
Good afternoon, good science folk.
I have been attempting to perform Westerns on serum that we've isolated from aortic punctures in rats, and have been getting a large glob ~ 40kDa with no apparent separation occurring after that (tested by Ponceau S) on both pre-cast (4-20%) gels and home-made (10%) gels. Our proteins of interest fall in this general vicinity, and I believe are being masked by this.
I've seen many many papers where people have run serum samples through SDS-PAGE, so I'm hoping that you may have some input as to how much sample to load (Do you dilute sample?), any special sample buffer formulations or modified PBS-T washings, etc.
Thank you for any and all input!
Your glob is probably albumin... it's the most abundant protein in serum...it's probably masking any of your other proteins. I think there's a albumin removal kit out there for designed serum electrophoresis, but I can't remember who makes it. Could also be immunoglobulins, too.
beckman-coulter sells a IgY based albumin removal kit that is supposed to be very specific and leave the rest of the proteins alone.
there may be similar kits from other companies.
the blue dye based kits (and others) will also remove some or all of the minor proteins.