can coommassie stained gel be western blot - (Jul/26/2012 )

Hi friends,
can i do western blooting of my proteins which are already coommassie stained, and if yes than how?

regards,
Binsan

It depends on the type of coomassie staining - the conventional acetic acid/methanol system fixes the protein so it won't transfer at all. I think colloidal coomassie, the type used in BN-PAGE will still work.

Colloidal coomasie is also commonly called G250, some solutions, like ours, do use a mild fix which will slightly reduce your transfer efficiency but it should still work. If you are using R250 (non-colloidal) there is an extensive fixing process that would greatly reduce the transfer efficiency. Zinc staining is probably the most compatible stain with western blot transfer.

my coommassie stain is comprised of coommassie brilliant blue in 95%ethanol, 85% phosphoric acid. the destaining solution is a mixture of ethanol and acetic acid. So it is colloidal coommassie. So can i go for western blot of my gel?

I'm a little confused on your stain, how can it be 95% ethanol and 85% phosphoric acid? (180%) Apart from that, if you are adding ethanol/methonol and an acid in a significant amount, at any step, that is going to "fix" your gel and will greatly reduce the ability to perform a transfer for a western blot. You can always try it, but expect to see poor results, if any results.

i have electroeluted coomassie r-250 stained proteins so transfer is, at least, theoretically possible.

first you have to remove traces of fixative (acetic acid is a somewhat reversible fixative). soak the gel in water.

then, use sds (up to 0.05%) in the transfer buffer to help resolubilize the protein.

it may still be inefficient but you should get some transferred.

a bigger concern will be the stain and how it effects post-transfer processing (some of the stain will be removed from the protein and should pass through the membrane).