WB - Wet - Markers not transferring? - (Jul/11/2007 )
I am having problems with my markers not transferring, my proteins are transferring off the gel onto the membrane fine, but the prestained markers are just disappearing. I've coomassied the gel after transfer and they aren't still there...
I've read some posts suggesting the proteins can transfer right through the membrane? has anyone got any experience of this?
It's got our postdoc scratching her head too, since she's done westerns for years with the same set-up and had no problems.
Cheers for any help!
Lee
I've read some posts suggesting the proteins can transfer right through the membrane? has anyone got any experience of this?
It's got our postdoc scratching her head too, since she's done westerns for years with the same set-up and had no problems.
Cheers for any help!
Lee
Hi,
this might be because of too long transfer period. possible reason could be that during transfer your marker may goes off mebrane, but if your protein size to too big that can not pass through pores of membrane so thats why it retains. if you are doing semi dry transfer 15 V for 18 to 20 min is fine.
good luck
have u tried to change markers and use another one? have u got the same problem?
how are u transfering?
are u sure that membrane and gel match perfectly and your ladder is not outside?
to be sure about your proteins put another membrane in the back of your first one so, if they go through, you will find them in the next one!
normally i transfer at 4*C O/N at 15-20V, but it's not a semi-dry.