I achieved uneven staining using silver stain on my SDS-PAGE gel, with my ladder (New England Biolabs #P7719S) staining the most, and a gradient down to my 5h timepoint staining least. To clarify, the leftmost well is the ladder, then 0h-5h timepoints sequentially.
I used pre-cast gels (BioRad #4561086), 10uL of sample (all from the same original sample, just different time points). I ran the stacking gel for approx 45 minutes on 50V and then for the resolving gel changed to 200V.
I followed the Pierce silver stain kit (Thermo fisher #24612) protocol.
Protocol available here:
https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0016358_2161478_PierceSilverStainKit_UG.pdf&title=VXNlciBHdWlkZTogUGllcmNlIFNpbHZlciBTdGFpbiBLaXQgVUc=
Thank you in advance.
I'm afraid this is just how it is with silver staining, with colors ranging from yellow to black, often the thicker bands appearing with a 'negative' central area and stronger color at the edges, as you see particularly on your marker track. Maybe someone else has a solution, but I don't know of any universal fix to this problem for complex samples.
I guess it is because of the higher concentration of the proteins (whether the ladder or the sample). Silver staining is usually done of the samples that has low concentrated protein that is unlikely to be detected by the Coomassie R-250 staining solution. Proteins samples not detected by the Coomassie staining solution are subjected to silver staining process. And as I can see in the picture, it is clear that the protein concentration is significantly high to be stained by silver staining process.
Shrawan Kumar Upadhyay I previously stained these using Coomassie stain and the concentration was too low to form bands. Is there an intermediate that should be used if your protein concentration is too low for Coomassie and too high for silver?
Thanks.
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