I’m facing the issue where my DNA protein is stuck near the wells in case of my EMSA native page. My protein’s theoretical pI is estimated to be 9.3. The pH of my 5x TBE running buffer is at around 8.3. Should I change the pH of my running buffer as well as the gel to get my protein DNA complex running through the gel matrix? The pH of my binding buffer lies at around 8.5. Should o change the pH of my binding buffer also?
What percentage of native PAGE do you use to run the DNA-protein complex, and do you use 0.5x TBE or 1x TBE as the running buffer?
I'm using 1x TBE as the running buffer. But I'm getting fuzzy bands instead of crisp bands Surajit Gandhi
I’m using 6% native page made with 0.5x TBE. I’m running the gel with 1x TBE at 80 V. I have changed the pH of my running buffer and gel to 9.5. Now the protein DNA complex are not stuck in the well. I’m getting the data but gels are not running properly. The bands seem fuzzy. I’ve attached the image here Surajit Gandhi
I am not sure, though. You can try changing the amount of protein and DNA used for the EMSA. Try using a smaller amount of protein for this experiment; Maybe this will solve the problem!
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