SDS-PAGE: transfer/blotting difficulty - (May/21/2006 )
Hi everyone,
I do westerns for a long time and because so far I have been working with a protein which expression is very high, I never had a problem BUT now, that I want to do an IP of a protein which is not very highly expressed, I am in trouble!
Before doing the IP I am optimizing lysis conditions, blotting and antibody concentration for detection...
I have encountered problems in the transfer.
Although a lot of protein is sucessfullly transfered into the membrane, by staining the gel with coomassie blue after transfer I realized that a significant amount of protein, not only high molecular weight proteins, stayed in the gel.
For eg. in my last experiment,
I loaded 20ug of total protein,
in a 12% mini gel 0.75cm thick).
I use a wet transfer system from Bio-Rad.
I did the blotting of two gels in the same apparatus at 20mA (~10V) for 16hours.
The blotting buffer :25mM Tris, 192mM glycine, 20% MeOH
The membrane is PVDF with 0.2um pore size.
Still didn't work! What should I do?
the good transfer is a crucial step because I am worried that I might be able to suceed in doing the IP and being able to transfer the IP protein and therefore fail.
Any suggestions?
A few thoughts -- try decreasing the MeOH content to 15% MeOH, or add 0.01% - 0.02% SDS to the transfer buffer, or increase the transfer time or current, or various combinations thereof...
How big is your protein? What kind of membrane are you using?
I know someone do western without transforming protein to membrane. Is there someone konw this IN GEL western and how to do that?
How big is your protein? What kind of membrane are you using?
Yes I read in Invitrogen website that is recommended for big proteins to include 0.01% SDS or increase current
The protein I am studying is 22kDa but the proteins that bind to it, are >40kDa.
That is why I have made a 12% gel. I might have to do a gradient gel to help in the transfer of the big proteins.
The membrane is PVDF 0.2um pore size.
Hi everyone,
You know what?
I found a solution to the low efficiency transfer.
I am runing total lysates on a 7.5% gel, this way the transfer of big proteins is optimum.
To check for smaller proteins I run a 12 or 15% gel.
I transfered 40V ON.