I am having problem with my proteins that have ~10 and ~11 pI and 17kD and 14kDa respectively. They do not get into the gel because of their charge. I tried purifying them even with a maltose binding protein tag to reduce the pI, however they are still too basic to migrate into the gel. My RNA is 130 nt long. I see the unbound RNA and decreasing concentration of unbound RNA with increasing protein concentration but the bands corresponding to RNA-protein complex are not very clear. Proteins alone are not showing up at all. I am using 5% Native PAGE. I am staining the RNA with Sybr Green and Protein with Sypro Red. Since I don't see clear complex bands, I cannot estimate the Kd. Any ideas on how to improve the quality of the bands and how to get the proteins to migrate into the gel instead of moving backwards? I tried non-ionic detergents such as Triton etc. but this did not help.
You can monitor protein/RNA interaction also using double filter binding assay.
MicroScale Thermophoresis (MST) is a powerful method to determine binding affinities. There are lot of publications available where this method has been employed to investigate protein-RNA interactions. The measurements are taking place free in solution, so you do not have to worry about the protein migrating into gels anymore!
http://www.nanotemper-technologies.com/applications/biomolecular-interactions/nucleic-acids/
Please also feel free to contact me directly for any further questions on this technique or if I can assist you with your MST experiments!
Hi Burak
I am in a similar situation trying to estimate Kd values for a basic protein (pI = 9.8) and its binding to different sequences (25-30 bp). Did you ever solve your problem how to design your EMSA, so you were able to determine Kd values? At the moment, I am only able to show a low extent of binding between the protein and the sequences even though the interaction between the protein and the sequences earlier have been shown to be in the order of ~5-80 nM.
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