Protein isolation from acrylamide gels - (Jan/26/2005 )
Hi, I would like to know if any of you have a protocol to isolate proteins from acrylamide gels. I want to extract proteins and then run them again for Western blot
What are you doing to the protein the first time round?
Coomassie staining or something else?
You can use coomassie to visualize in order to cut out your band, but avoid using methanol and acetic acid during the stain or destain procedure - this will fix the protein into the gel and reduce the yield during protein elution. Use water as the solvent instead...
I think you have 2 options for elution
1 - electroelution
example : http://www.fermentas.com/catalog/kits/kitelutatubet.htm
2 - ultrafiltration
someone might be able to give a "from scratch" protocol though. Im a fan of kits...