When I stain my SDS-PAGE gel with silver-ammonia staining I get an unstained or lightly stained area just above 75 kDa band in all the lanes of my gel. I use a 12% resolving gel. Can anyone help with this issue? I have tried using fresh buffers and solutions. The running buffer I use is tris-glycine. I have attached an image with the described area circled.
Your gel picture is great and you don't have to worry about this particular area (circled). This observation just means that not much proteins are present in this area.
I have not tried with coomassie staining since the amount of protein I get is very less in every preparation. I cast my own gels.
I know that there are bands in that region because previous records from our lab have the bands. I have been following the same protocol, but for some reason I am facing this problem. I am unable to pinpoint exactly at what step I am doing the mistake.
Thanks Oliver.
I do pour off the butanol and wash with distilled water before couple of times and then dry it with whatman paper, before pouring the stacking gel. As you mentioned it happens in the ladder too. I'm looking at the entire protein profile, so it is important to sort out this problem.
Dear Rahul,
Do you get the solution for your problem? Currently I got some problem with you. I could not get the bands >100 kDa (it was a 'blank' above 100 kDa), but previously it was no trouble using the same technique.
please let me know if you have found the troubleshooting. Thank you very much.
Hi Yonika
Yes, I found the solution for the problem. Now before fixing the gel I wash it with Mili q for 10 mins and then fix it in 50% methanol. Then I proceed as usual.
Sorry, for the late response.
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