5 replies to this topic

[pakbiochemist]

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

regards,
Binsan

[bob1]

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.

[proteaMatt]

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.

[pakbiochemist]

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?

[proteaMatt]

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.

Edited by proteaMatt, 27 July 2012 - 12:04 PM.

[mdfenko]

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).