Hi,
I am doing a gel shift assay to confirm that my recombinant protein will bind to DNA in vitro. I am using my rBAF180 and miniprepped pET30 vectors. I have tried undigested and digested DNA.
The results I am getting are somewhat puzzling. I know the concept is that DNA:protein complexes migrate slower than free DNA, but what I seem to be finding is that the DNA:protein complex is migrating faster. I've looked it up and it appears (according to Fisher website) that if the DNA is circular, the DNA:protein complex can move faster than free DNA.
The first gel I attached is the undigested DNA (in this case pET30b). The second and third gels are EcoRI digested pET30a/b (but it is possible that these digests were not complete). Each of these experiments were set up individually and all used different DNA minipreps.
Can anyone help me with the interpretation of these results? I think that the DNA must be circular and the protein bound to it is migrating faster. Does anyone have any different suggestions?
Thank you!
Consider leaving out the loading dyes from your sample buffer, since they are causing the dark areas on the gels. There could be bands there, but they can't be seen.
A possible artifactual reason for the DNA to run faster in the presence of the protein is that the protein is contaminated with DNase, which is cleaving the DNA into smaller pieces.
Consider leaving out the loading dyes from your sample buffer, since they are causing the dark areas on the gels. There could be bands there, but they can't be seen.
A possible artifactual reason for the DNA to run faster in the presence of the protein is that the protein is contaminated with DNase, which is cleaving the DNA into smaller pieces.
Hi there,
About completion of digest: there should be a single band which corresponds to the size of the plasmid according to the DNA ladder. Is it the case?(As you are not mentioning if one of the lane corresponds to DNA alone...)
I agree with leaving the dye out. You can use dye-free loading buffers and observe the difference in Index of refraction by eye to visualize loading. Ficoll based loading buffers are particularly easy to visualize this way. As an alternative, there are some fast-migrating loading dyes (orange G I think), that may not obscure the bands of interest (https://www.neb.com/products/b7022-gel-loading-dye-orange-6x)
I also agree that there may be DNAse contamination. The faster migration may be consistent with digestion of the dsDNA. The gels you are showing are not fully labeled. I assume the left lane is DNA with no protein and the left lane is DNA+Protein. If so, the migration pattern of the DNA only lane looks odd. The DNA is not forming a discreet and narrow band, as would be expected for linear DNA. Does the apparent mobility of the DNA match the expected mass? If not, you may want to optimize the digestion and clean the digested DNA before using it in the EMSA.
For these sorts of Agarose EMSAs, I found that using 1/2xTBE and keeping the gel/buffer cold (4*C) seemed to improve resolution, though it wasn't really necessary for detecting binding to large substrates. Running a PAGE emsa with a smaller dsDNA target might show the binding more clearly, if such a modification is feasible given your reagents (e.g. some protein/DNA complexes are immobile in PAGE due to unusual structures).
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