Hello all!
I am struggling to understand why my bis-tris gel is not resolving well. There is not a uniform band forming in the lanes causing the gel to turn completely blue. I am using this recipe from Indiana university: https://medicine.iu.edu/faculty-labs/corson/protocols/gel-recipes
My samples are prepared in 3x SDS sample buffer. We have used LDS sample buffer before and found that SDS sample buffer is easier to use and doesn't affect the results.
Previously, I have been successful using this recipe so I am not sure why all of a sudden it has stopped working.
Recently, I remade the 3.5x bis-tris buffer and the MOPS buffer. I have also tried redoing LDS prepared samples on the gels made with the new buffers and have gotten the same results. So I believe it has to do something with the 3.5x bis-tris buffer but I cannot put my finger on exactly what I changed between the first time making the buffer and now. Any further suggestions would be greatly appreciated!
Hi Kimberly Edicha,
As I can see, your protein bands have not resolved at all. Also, the stained gel seems to be smeary. I have faced a similar issue in the past. But, there can be multiple reasons, including a change in the pH of the stacking (6.8) and resolving (8.8) buffer. Firstly, I suggest that you check the pH of your buffers. If they are not the same, try preparing a fresh buffer for your gels. At times, microbial contamination can lead to a shift in the pH of these buffers. So, you can get them autoclaved to use for longer periods.
Secondly, I can see that the layer of stacking gel is too small. This may be a reason why your proteins did not resolve. Ensure that your stacking gel is in an adequate proportion (at least 1 cm) in your SDS gel. This may also happen at times if your SDS gel is not properly polymerized. One should give enough time for the gel to polymerize. The protein concentration does not seem to be high. Does your sample have a high salt concentration or any organic solvents in them? Hope that you processed your protein samples by heating at 95 or 100 degrees for 5-10 mins after the addition of loading dye?
Hope this helps you. Good luck!
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