Hello, I wonder if I can stain nitrocellulose membrane with Ponceau S but not directly after transfer as it's usual do, but after staining with both antibodies (primary and secondary) and development the membrane (chemiluminescence). Is anyone use Ponceau S like that? I will be grateful for any suggestions.
Hi tere
Of course you can stain your membrane after probing but the protein profiles obtained will be affected by the binding of primary and secondary antibodies.
Ponseau S stains total protein nonspecifically, including antibodies.
Although the amount of protein content contributed by antibodies is expected to be very low (after wash) and negligible, it cannot be taken for granted, for methodological reasons.
It is better to stain Ponseau S, obtain a quick scan record, destain and move on to antibody staining.
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If you really want to try your method, you can first do Ponseau S one time (followed by washing off) before antibody staining, and then do Ponseau S a second time, and compare the two times' results.
We always stain our blots with Ponceau S after antibody staining and X-ray film exposure (ECL system). Ponceau S stained blots are useful for comparing the protein profile with bands on developed film. I agree with Liger that the protein profile will be affected. The targeted protein bands will be more intense due to binding of antibodies.
We always do the Ponceau S before blocking the membrane with BSA or milk which will be . The bands are fainter if you do it after the membrane protein saturation. Also, as Panduranga said the antibody will detected and will increase your band, not sure if you could then compare the intensity of bands.
uptill which stage should i wash the Nc membrane after staining with Ponceau S , ? and is deionized grade-C water is enough to wash?
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