Hi everyone,
Yes, I know, almost anyone nowadays is using radioactive labeled probes for EMSA, but since I was not lucky with fluorescent labeled probes I tried to do it old way. Can anyone please suggest me how to improve the quality of my gel shifts? The bands look pretty diffused. I am pretty sure my experiment is working but I wish I could have nicer pictures.
I am running 5% AA/0,5x TBE gel in 0,5x TBE for 2h at 150V and after dry the gel with Biorad gel drier and vacuum.
Any suggestions please?
We load sample in long thin tip that can load sample directly on bottom of well. Also there is 10% glycerol in reaction buffer to increase the density of reaction mixture so that it can be settled on the bottom. Also have you monitor the temperature of the gel, you need to keep your gel cool.
Hi there,
To go further in the above contribution's way, if possible try also to diminish the loading volume and to avoid overheating during running you might lower the voltage. You could also consider increasing polyacrylamide concentration.
Thank you very much for suggestions, I already use 12,5 ul volume for the reaction. I will keep eye on the temperature even if I was thinking the gel does not get overheated. I will try higher AA concentration. Do you think the prerunning might be also an issue?
Long loading tip works wonders, try it! Try to load stuff on the well bottom, it makes a huge difference!
Are you using developing cassette with the screen? We had to purchase the new one since the old screen was in bad shape. PM me if you need more details, good luck!
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