Colloidal Coomassie - protocol for mass spec analysis (Feb/26/2008 )
I want to use colloidal coomassie to visualize proteins I have IPed and then analyze specific bands by mass spec. I've looked around and found a few different descriptions as to how to use colloidal coomassie, and am a little confused.
My understanding is that when you do a silver stain you fix the protein into the gel, which prevents any useful MS analysis down the road. About half the protocols I've seen for colloidal coomassie involve a fixation step in acetic acid and methanol, wouldn't this have basically the same effect as the silver stain. It seems to me this fixation step would prevent me from doing MS analysis later. Is this step required for effective staining? Or is it more of a user preference?
Also, is colloidal coomassie re-useable like standard coomassie stain, or is it one-time use only? Are there any special disposal requirements? Do you simply destain with water?
Finally, is there a specific recipe and/or protocol that works the best, at least for my purposes (staining and subsequent MS analysis of bands)?
Thanks for any help!
Some silver staining protocols involve a cross-linking step with glutaldehyde in which protein is chemically cross-linked. Most coomassie blue staining protocol uses acid/MeOH induced denaturation or physical cross-linking, thus is reversible.
No particular disposal protocol is required.
colloidal CBB give better results than convential CBB; it is compatible with MS; it is reusable