Help needed-Protein extraction from native gel - (May/18/2006 )

Hi everyone,

I would appreciate any protocol for the extraction of active proteins from a band on a native gel. I need to see which one of my bands has the reduction property I am looking for (I have about 6-7 bands right now). After that it'd be sent to mass-spec.
By the way- would it be wise to stain in with coomassie blue, or should I cut it without staining (learning about the position of the band from another gel....).

Thanks a lot,
Boaz.

what we do is cut a strip from the gel and stain (and destain) it, align it with the rest of the gel and cut our band of interest from the unstained portion. we extract the protein by homogenizing the gel in buffer, pelleting the gel and collecting the supernate. you can also electroelute the protein from the gel cuttings.

if you stain your protein then you will probably lose its activity. it may be okay to stain for ms.

Thanks for the advice. My Prof said something about extracting it into a buffer, what I had missing was the homogenizer part most likely. I'll try that, thanks again.

some people extract by just incubating the gel slices in buffer without homogenizing. the protein will leach out of the gel. we homogenize because it is faster.