I am attempting to perform an EMSA with a transcription factor and its wildtype binding sequence but the first attempt showed that the protein never left the well. After some research, I have discovered that the theoretical pI of the protein is approximately 8.8 and my running buffer is 8.3.
What is the best way to run an EMSA for this protein? I am worried that if I change the loading buffer pH that the protein:DNA binding might be affected. Can I just adjust the pH of the running buffer to 1 point above the protein's pI (e.g. 9.8) with NaOH? Do I need to adjust the pH of the gel as well?
Hi Jiejiao Liu
I was able to get the protein to migrate through a NATIVE PAGE gel by using a higher pH (I had to go up to pH 11/12). However, when I tried to do the binding assay, no DNA was bound by the protein. I adjusted the pH of TBE with NaOH, which probably meant there was too much salt in the buffer for DNA-protein binding. Therefore, I would suggest if you are going to try an increase/decrease in pH to use a more appropriate buffer. Let me know if you get it working, as I still haven't!
Jiejiao Liu
Also, you can try lowering the pH and switching the electrodes so that the protein still migrates down instead of up and out of the wells
Jiejiao Liu
https://www.biorxiv.org/content/10.1101/825679v1.full.pdfThis might be helpful
Jiejiao Liu
I don't fully understand your results. Feel free to post a photo of the results and I can have a look- Badges
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