I'm running an EMSA to show the DNA binding activity of recombinant proteins versus the Wild type. Previous assays run by my research group using a different test system show the protein-DNA complex migrating about halfway into the gel, however my complex samples seem to get stuck just bellow the wells, whereas the free DNA migrates to the bottom. Also, my cold probe lane shows an unusual band halfway down the gel, despite having increased the concentration to 1000-fold molar excess. I've tried multiple concentrations of protein and labeled probe with similar results. I'm using 5% polyacylamide TBE gels; 0,5X TBE running buffer; and the binding buffer is 10 mM Tris-HCl, pH7.5, 50 mM KCl, 1 mM DTT, 5% glycerol. The probe is 30bp DNA labeled with biotin. I incubate the protein with cold probe for 30 min (increasing to 1 hour did not make a difference), and with labeled probe for 20 min. The protein is 11.6 kDa and has a basic isoelectric point of 11. I run the system on ice, applying 100 V for 45-60 min.
The migration speed of the protein-nucleic acid complex depends on your surrounding pH.
Since your calculated pI is 11.6 and the buffers pH is 7.5 your protein should be positively charged (in contrast to the negatively charged DNA). Thus i am wondering that the complex is even entering the gel. I would suggest you to try a higher pH in order to increase the negative charge on the recombinant protein.
Also: be cautious that the pH of Tris is temperature dependent. (see https://international.neb.com/tools-and-resources/usage-guidelines/ph-vs-temperature-for-tris-buffer) Your buffer won´t be at pH7.5 on ice, if you equilibrated at room temperature before.
Best of luck,
David
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