Hi there,
I'm doing western blot recently. My SDS-PAGE gel was first stained with the Coomassie Blue G250 and then after disstaining I transferred it to a PVDF membrane. After the transmembrane process, there are some blue bands could be observe on the VDF membrane which might owing to the incompletely disstaining. The result was visualized by HRP+ECL plus and it worked in my experiment. I'm just wondering: will Coomassie-Blue-stain protein affect the result or, put into another word, can CB-stain protein be detected by ECL plus channel?
Thanks,
Guangqi
Coomassie won't get picked up by ECL. The HRP-ECL reaction creates visible light a ways away from the red range. You can see this if you take some HRP and toss it into an ECL mix.
HOWEVER, if you want to do NIR on your blot, you've made your membrane pretty crummy since coomassie is unbelievably bright in the 680nm range. Even blue ladder is blinding.
I'd be kind of doubtful if antibodies would even bind to your membrane since you've got a bunch of other molecules stuck to various parts of them and it might be blocking something the antibody recognizes. Usually, you should transfer and then do staining to check for transfer efficiency.
Then again, someone else asked this question too, so it's possible depending on what you did! https://www.researchgate.net/post/Is_western_blotting_with_comassie-stained_acrylamide_gels_possible
You can always add TCE (2,2,2-Trichloroethanol) to your gel, 90µl per gel only in the resolving part. After running you can visualise proteins under UV light with ~30 sec exposure and than do the transfer. The TCE binds to the tryptophan ring which causes it to light up under UV light. It does not reduce western blotting conditions or antibody binding. After the transfer you can even visualize your proteins in the blot and see how much has transferred to the membrane.
http://www.sciencedirect.com/science/article/pii/S0003269703008017
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