Problems with HMW proteins and Western blot - (Nov/20/2007 )
Hey there. I've been having a couple problems which reoccur when I look for high molecular weight proteins with Western. First, I get heavy background on my membrane at HMWs. The blot looks great below 100kD, but at higher MW there is significant background. I've tried lowering my antibody concentrations, increasing my washes, but no change. It wouldn't be problem if my protein of interest wasn't 250kD. Second, does anyone out there look at HMW protein expression and if so, do you use PVDF membranes and boil your samples before loading. I use mini-gels, 12 wells and load ~25ug total protein per well, run the gels at ~30mA and transfer at room temp overnight at 15V. Thanks.
fmusc
fmusc
good resolution of HMW polypeptides is achieved by gardient gels
I routinely do western blots for detecting 220kD protein.
I use PVDF membrane and boil my samples before loading.
I suspect that background may be antibody problem. Some times i faced that when i used antibody from a company.
They recommended me going for overnight blocking in TBS.
Hope this helps.
I use PVDF membrane and boil my samples before loading.
I suspect that background may be antibody problem. Some times i faced that when i used antibody from a company.
They recommended me going for overnight blocking in TBS.
Hope this helps.
Thanks for the advice. In repsonse to The Bearer, I forgot to mention that I am using gradient gels. In response to newarray, do you get any protein aggregation after boiling your sample? My protein of interest is a membrane bound protein and boiling appears to result in the aggregation of these proteins. Also, how do you transfer to PVDF? Overnight, 4 degrees, room temp. etc. Thanks so much.
Hi,
One of my proteins is at >250 kD. It's aggregate prone and will not move to separating gel when boiled. I'm not using gradient, but managed to get the band when I heat the lysate in 1X Laemmli buffer at 65C, 15'. Cell lysate was prepared in RIPA with Triton X-100.
I run gel at 150V and transfer at 100V with cooling. Tried lowering run voltage and transfer voltage (o/n). Both gave similar results.
Used PVDF membrane.
...-...
One of my proteins is at >250 kD. It's aggregate prone and will not move to separating gel when boiled. I'm not using gradient, but managed to get the band when I heat the lysate in 1X Laemmli buffer at 65C, 15'. Cell lysate was prepared in RIPA with Triton X-100.
I run gel at 150V and transfer at 100V with cooling. Tried lowering run voltage and transfer voltage (o/n). Both gave similar results.
Used PVDF membrane.
...-...
Thanks Biowizard. That helps a ton.