I have a protein whose function is to reduce transcription factors. To check whether my protein is active I have to do DNA-EMSA. I am using my purified protein to reduce TF from nuclear extract from Hela cells. To do so, I am using fluorescent labelled 23 bp oligonucleotides which were annealed. EMSA was performed by binding the probe with a nuclear extract (5ug) and running samples in 6% gel using 0.5X TBE buffer (44 mM Tris, 60 mM boric acid, 1 mM EDTA).My reaction contained 4 μl of 5X binding buffer(50%glycerol, 75mM KCl, 25mM MgCl2, 50mM Tris pH 8),5 μg Nuclear extract and my recombinant protein incubated on ice for 30 min .After which 0.5 μl of 1 µg/µl poly(dI-dC) was added and incubated for 5 mins. After this 1 µl (10 pmol) labeled probe DNA was added and reaction was carried out for 30 min on ice.Reaction volume was 20 µl. My protein does not bind to the DNA probe. I am getting a smear band and not a crisp sharp band. If I can get a good band then it would be better.What I can change to get the good results.
Thanks in Advance
TTIWWOP. This thread is worthless without pics.
It might be helpful to see an image of the EMSA with and without protein. You may need to optimize your DNA/Protein ratio, and you may need to play around with buffer and incubation conditions extensively before the protocol will work well (especially if you're doing this without other publications to guide you.
A smear in an EMSA may indicate that you have multiple Protein:DNA binding ratios (e.g. on some DNA's you have one protein bound, on others you have 2, etc...).
Alternately, the proteins that bind to the DNA may be becoming gradually unbound during the separation. This would tend to accelerate the migration of parts of the band, widening it. Once the separation is started the binding equilibrium is disturbed, because if/when the proteins dissociate from the DNA, they will end up physically separated from the DNA due to the ongoing migration. This may be a problem if your proteins have an appreciable Koff rate.
You could try to 'fix' the proteins onto the DNA using any number of fixatives, but that could turn into a nightmare (fixatives sometimes have 'weird' effects on migration).
When I was running an EMSA using a Cy3-Labeled 90nt ssDNA oligo and TRF2 protein I found that 1/2x TBE and a 1% Agarose Mini-Gel run at 4*C at 100V gave me the best resolution of the bands. It ran fast (~0.5hrs), and gave adequate resolution of the oligo and the super-shifted species.
(http://www.ncbi.nlm.nih.gov/pubmed/25115914)
@Brian Thanks for the suggestion.
Yes my DNA probe is for AP-1 which binds to the homodimer/heterodimer. so it possible that for some DNA homodimer is attatched while to others heterodimer.I have attached ppt which has the image. see if that is helping for any suggestion.
Also, how did you use 1% agarose gel does it allow protein to pass through. Do you use the same Agarose protocol for it?
@Komal
It does look like you've probably got a super-shift, but it might take some optimization to make it quantifiable and publishable.
I used the same grade of agarose that is routinely used for DNA detection and quantification; it wasn't any kind of fancy low melting point agarose. Protein/DNA complexes generally migrate well in agarose (can be confirmed using E. coli SSB or T4 Gene 32 Protein available commercially). However, small DNAs don't usually resolve well in Agarose. You won't get a discrete band for an oligonucleotide, but you may still get a quantifiable (if 'fuzzy') band, as I did.
My agarose EMSAs included one protein-binding EMSA and a displacement-loop EMSA (D-Loop). The protein binding EMSAs are similar to what you're looking at. The nature of the supershift varies with the characteristics of the proteins. For instance in my work I found that migration of TRF2/DNA complexes was severely retarded in agarose electrophoresis (they stick in the wells at high enough [TRF2], which has been previosuly published). In contrast, TRF1/DNA complexes did migrate into the gel under generally comparable conditions. I also ran a competition experiment, that you may want to do in the future once your EMSA is working well. (See below)
You might consider switching to polyacrylamide gels, which have superior resolution capabilities for EMSAs.
I think Komal is using a 6% 1/2xTBE polyacrylamide gel.
My experiments required an agarose gel due to the nature of the proteins and DNAs I was studying. A complex of TRF2 bound to a 90mer of telomeric DNA is essentially immobile in polyacrylamide, and I wanted to use the 90mer for the EMSA since it was the same template used in the D-loop assay. Additionally, the D-loop EMSA required using agarose since it needed to separate both a plasmid (also essentially immobile in polyacrlmide) and the oligo in the same gel.
The protein binding EMSA was adapted from the D-loop EMSA in the interest of ensuring the results were directly comparable, and not reinventing the wheel.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques