I am working with cyanobacteria and to study changes at photosystems level I have performed BN-PAGE. But unlike the results I saw in other papers I do not get the images? So I am confused whether should I stain the protein or increase the loading concentration of protein ?
In BN-PAGE, staining (such as with Coomassie Brilliant Blue) is typically required to visualize the protein bands. Without staining, you won’t be able to see the results. Here are some points to consider:
You need to stain BN-PAGE gels to be able to see the bands on the gel. While some highly abundant proteins may be visible immediately after electrophoresis as they've bound so much of the Coomassie Brilliant Blue in the cathode buffer that they are visible over the blue background of the gel, this is not typical for most proteins.
The easiest way is to stain with Coomassie Brilliant Blue. Depending on the concentration of coomassie G-250 that you used for the cathode buffer during the run, it may be possible to fix and destain the gel so that the bands will become visible from the coomassie bound to them in the gel. For better sensitivity though, coomassie G-250 or R-250 stain can also be used. If you look at this link, it gives several potential methods. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/nativepage_man.pdf#page=26 While these are for commercial pre-cast bis-tris blue native page gels, the methods should be broadly applicable to other BN-PAGE gel types.
Silver staining is another option, but it is a lot of time/work.You need to pre-plan to use it, as otherwise it is all too easy to overload the gel due to the exceptional sensitivity and you also have to use a reduced amount of coomassie in the cathode buffer.
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