Western for a 500kda protein - some troubles, need help! - (Jul/18/2008 )
Hi, any help on this topics would be appreciated greatly as i am really having a hard time getting this to work.
So I am trying to resolve a 500kda protein on a western.
-Separating: 6% tris-glycine (polyacrylamide)
-Ran overnight at 40V (16hrs)
-Transfer for 2h at 120V, 10% methanol tris-glycine transfer buffer
Nitrocellulose transfer membrane
The problem is either that the protein is running off the gel or it's not transferring at all, or possibly diffusing during running. I get no bands on my western, I've repeated this about 3 times.
Can different gels work better for large proteins? Are there any methods you know of to resolve large proteins efficiently on westerns? Thanks for any help, much appreciated!
You can try using a gradient gel such as a 4-20% tris-glycine precast gel. That'll help from keeping the protein from running off. Also for the transfer, is there any SDS in the transfer buffer? In my experience huge proteins have problems moving out the the gel, especially if its thick so I usually add about 10 ml of 10% SDS solution into 1L of my transfer buffer. Adding too much SDS can cause the protein to not stick to the membrane however. Also how are you transferring? Are you doing a wet transfer? We usually have a 20% methanol tris-glycine buffer... the high methanol content seems to help proteins stick to the membrane better.
Also have you thought of resolving the proteins on an thin agarose gel? that can work for resolving extremely large proteins.