Hello fellows,
I am doing EMSA. I wanted to check for the binding and efficacy of DNA and nuclear extract proteins. So, I run Acrylamide gel (native, 5% , 0.5xTBE buffer, with DNA view dye ).
I took image for the gel, and as we can in the attached image, the DNA is higher than DNA/protein.
So my question : Is this a true signal ? why I see this phenomena ?
To me it looks like your DNA has not interacted with any of your proteins/samples. Pure DNA and a mixture of DNA/protein will not have the exact same mobility. The difference in migration is likely due to the presence of protein in your sample, you can also see that the band are not straight where you have added the nuclear extracts.
It really looks like no shift. As judged from your gel there is no positive control. Thus, you cannot tell whether something wrong happened during the experiment or whether your DNA does not interact with your proteins of interest.
Hi there,
Is the DNA only sample prepared exactly the same way as DNA+prot samples (apart from the absence of protein)? The bands have very different shape indeed. As a control I would prefer DNA + non interacting protein (BSA for instance) and DNA + nuclear extract without the protein of interest.
Actually the reason I use EMSA kit components as control is that I tested it before and I can clearly see the DNA shift on the nylon membrane.
But here, when I ran the gel only and took image ( and not transferring to the membrane ) I see this abnormal behavior. Even if there is something wrong with my samples , what happened with the kit samples ? I did it twice and the same results.
Which lysis buffer are you using? Is it self prepared or kit? If it is self prepared, prepare it again fresh and add 1mM EDTA (final conc.) in the lysis buffer. Your DNA+Protein solution must contain EDTA.
The only possible problem I can see is DNase activity. You can prepare your gel components freshly. And in the worst case another problem could be is your DNA competitor. Check what type of competitor you use. Check its molecular wight. Check whether it is degraded and binds to your protein and gives nonspecific band. This is a rare case. You can incubate DNA and protein without competitor to check this problem. I never had problem even without competitor. Check the image (first lane is free DNA). So you can try.
~30-40p. Purity means whether it contains cytoplasmic fraction. Right? I run western and probe with a cytoplasmic protein like GAPDH and nuclear protein like liminin or histon. A littile contamination is not a problem.
if circular DNA is used (e.g., mini-circles of 200–400 bp), the protein–DNA complex may actually migrate faster than the free DNA, similar to what is observed when supercoiled DNA is compared to nicked or linear plasmid DNA during electrophoresis.
Protein -especially crude extracts will change the mobility because of salt , and crowding effects. See that the bands show a meniscus that is not explained by being a lot of DNA there. The experiment has to be repeated in a more convincing way. From he looks of it however I guess that either there is no protein or no interaction. If the proTein is OK and the interaction is known, I conclude that the labeling is no good or that free biotin of streptavidin blocks the interaction.
Dear Dr.Joseph ,
Thank you for ur guidance.
Actually I checked the protein by western ( using 15 ug conc.) and I can detect the band very clearly.
And my labeling efficiency according to the kit is around 50-60% ,
How to make sure that the free biotin of streptavidin is the problem ?
And what is the the max. range for DNA and protein concentration I can use in EMSA ? I know standard protocols said it's up to 10 ug for protein,
I wonder if i can increase the amount of protein more than that ? and also amount of DNA ?
Thank u
Check conditions from the "Thermo diagnostics" kit. I s the photo a chemiluminecence image or DNA stain? Is your biotin at the ends only?
Use a DNA aliquot to see if its bound to streptavidin beads and therefore good for binding . 10 μg pure protein is a lot relative to ng of DNA you are using. Perhaps you can increase the amount of DNA not the protein .
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