what is the appropriate SDS-PAGE concentartion to be used? - (Dec/07/2005 )
Dear All
I would like to separate 3 proteins of molecular weights of 49, 45 and 43 KDa on SDS-PAGE for westren plotting on PVFD membrane. what is the appropriate SDS-PAGE concentartion to be used to get those proteins separated from each other with a good distance, allowing cutting of the PVFD memebrane into 3 pieces each contain only one protein??
I tried 10% SDS-PAGE, but I had to do membrane stripping 2 times in a row, and the signals obtained after stripping were always weak. So I want to avoid that stripping process from the begining.
thanks in advance.
I would like to separate 3 proteins of molecular weights of 49, 45 and 43 KDa on SDS-PAGE for westren plotting on PVFD membrane. what is the appropriate SDS-PAGE concentartion to be used to get those proteins separated from each other with a good distance, allowing cutting of the PVFD memebrane into 3 pieces each contain only one protein??
I tried 10% SDS-PAGE, but I had to do membrane stripping 2 times in a row, and the signals obtained after stripping were always weak. So I want to avoid that stripping process from the begining.
thanks in advance.
Run a longer gel with 12% SDS-PAGE, so that there is a bigger separate among the 3 proteins. When transferring to the PVFD, cut off the top part of the gel without your requires proteins to reduce the size of the blot.
I hope this may help.
I would use a gradient gel. Perhaps 10-25%. Run it real long, hope for the best.
No you do not want to use a gradient gel to try to separate proteins of similar weight.
This is because a gradient gel will compress the total range ie you will "catch" more of the larger proteins on the same gel with smaller proteins ie they are not being resolved, they are actually migrating closer together.
Gradient gels are good for screening so that you can get a greater molecular weight range on the same gel
This is not what you want to do here.
You want to choose a single gel concentration that is appropriate for resolving proteins at the 50 kD size. That way larger proteins are slowed down and smaller ones are sped up.
As was suggested, a 12% gel would be just about right for this.
Getting longer plates so that you can run a longer gel so they resolve more is also a great suggestion.