Protocolpedia - The Encyclopedia Of Lab Protocols

hi all,

i'm currently being frustrated by trying to run my protein through a semi-dry blotting system.... the extracted cell protein (i'm actually looking for UCP-2, 31kDa) is eletrophoresing very weel (as demonstrated by coomassie) but i haven't managed to get it to transfer yet.

i've tried both continuous and discontinuous buffer systems, and a range of power settings (15V for 30m/1h..... 150mA for 1h.....250mA for 1h....mA per cm2 of gel for 1 h) and none of them have caused protein to show up on ponceau s staining....

i'm using sigma's semi-dry western blotter if that helps

where am i going wrong?

Thanks for the help....

i'm using a nitrocellulose membrane, and have now managed to partially transfer the protein by adding sds to the transfer buffer, still bot 100% though!!! am running at 5V for 90 minutes

UCP-2 is a highly basic proteins, so it should be transfered easily and quickly. check your semi-dry system to see whether other proteins (just like marker) are transfered. also check ponsinus staining of your protein, this can be done by dot the purified protein on membrane then check. if it doesn't work, you can use fast green. also, put two membranes on both sides of the gel during transfering, and stain the gel after.

i'm using a nitrocellulose membrane, and have now managed to partially transfer the protein by adding sds to the transfer buffer, still bot 100% though!!! am running at 5V for 90 minutes