SDS gel troubleshoot - My proteins do not show up in the gel. (Aug/27/2004 )
I am running an SDS gel for 2 hours at 200v 500mA in a precast gel , my protein is the transporter ABCG2, i am using the STOP loading buffer, it seems that the proteins run off the gel but when i did the coomassie staining and the transfer to nitrocellulose i can not even see a trace. I did this experiment once and i found bands. Can anybody try to give me an explanation. Thanks , any help will be appreciated. I am a first year graduate student in biochemistry.
I'm not really sure if I understand your method,.. but 2h at 200V sounds alot to me, but then again it depends on the Mw and the precentage of gel. I used to run 10% precast at 1h 200V (Biorad system), and that gives a nice spread on the 50-150 kDa, so if ABCG2 is in that range, try that (=shorter running). Or run prestained markers so you can follow the Mw while the gel is running.