Hi, community! I have been using 12% SDS gel to separate my protein of 57kda since last year and my SDS was working just fine. Lately I have been struggling as my protein is not entering the separation gel anymore. Everytime I run my gel the marker and the loading buffer seems to be moving just fine and reach the end of the gel, but when I stain nothing appears on the wells where I loaded my protein. When I use a precast gel the protein bands appears. I have tried using new 30% acrylamide and redoing the buffers but still no results. What could be happening with my gel? Any ideas?
This is most likely a buffer problem (proteins are not charged negatively). Remake your gel buffers and make sure the pH is ok.
4× upper buffer mix Tris-HCl/SDS pH 6.8 (0.5 M Tris, 0.4% SDS in water)
4× lower buffer Tris-HCl/SDS pH 8.8 (1.5 M Tris, 0.4% SDS in water)
As commercial gels work fine this excludes problems with the loading buffer, running buffer, and the electrophoresis itself meaning that the problem is in the casted gels.
I would agree that the buffers may be the problem; I've found in the past that extra care has to be taken particularly when making the resolving buffer (1.5M Tris-HCl, pH 8.8) as its pH can slowly creep up or down (depending on if you used Tris base or Tris-HCl to make it) after you've initially adjusted it. After adjusting to pH 8.8, I would let it sit on a stir plate for a couple hours, then re-check and re-adjust it; you could do this a couple of times to be sure.
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