I am doing EMSA for the identification of transcription factors. I am using total nuclear proteins. When I run the sample of Protein alone and protein plus DNA sample there is no difference in location of band. What is the reason? The probe size I am using is 250bp but at the bottom I observed a decrease in probe concentration.
Probably because the protein extract contains many proteins that will bind the DNA in different ways and since they have different molecular weight only very few copies of the DNA are shifted with a particular protein. So rather than having a single signal corresponding to a specific binding (of even a ladder type), you're having some kind of unspecific shift and therefore a "smear" of DNA that can hardly be detected using sybr or EtBr. If you were using radio labelled DNA you might be seeing this smear.
the other hypothesis is that your extract contains some DNases that degrade you DNA....
I am going to try my first EMSA with IR dye. I think you would need to isolate specific TFs instead of just all nuclear protein. Also I have found it is always best to have end labeled probes because anything that binds directly to the DNA can disrupt the TF binding or if it does bind the protein could limit the signal from the stain...
Maybe you could try to increase the amount of poly-dIdC added to the binding reaction, just in case you have many proteins in your extract that are able to bind unspecifically to your probe (if that is the reason for observing a decrease in free probe but no bands). Depending on the interaction, you can see differences in the binding affinity due to temperature. In my experience, I was unable to observe any interaction for my protein-DNA system at 4 degrees (which is usually recommended to stabilize weak interactions), but the interaction looked great at room temperature (and it was specific, according to competition experiments). You can also try a very general binding site as a control, just to be sure your protein extract is working properly. I don't know what is the organism, but for plants and fungi a G box (CACGTG) would work perfectly, since there are many G-box binding factors and you should observe binding to this probe.
Also try different binding buffers, some include polyamines or different ionic strength, which could help stabilize some interactions....
Good luck!!
DR.MAY AL-DOORI/
Electrophoretic Mibility-Shift Assay with SYBR Green &SYPRO Rubi EMS .This fluorescence -based electrophoretic mobility shift assay provides a fast and quantitative method to detect both nucleic acids and protein in the same gel.There is a difference in the time required to separate both ,, Staining only takes 20 minutes for nucleic acids and then about four hours for the subsequent protein staining.
I do suggest to try InVitrogen Kit.,, I worked alot with Plasmids and did get experience with protein (s) separation & staining in collaboration.THANK YOU
Thank u so much for your valuable suggestions. There is one band at top and one at bottom when stained with SYBR Green . It means band at bottom is of free DNA and band at top is that which is attched to protein? I have loaded only protein too in one well. In that case ther eis weak signal at top with SYBR Green and no bottom band. I think some of the DNA is restricted to top by DNA binding proteins because there is decrease in bottom band intensity with increasing protein concentration.
Dr. May Al-Doori you have done Flourescence EMSa with SYBR Green and SYPRO Ruby. Are you using any non-competitive inhibitor? and what binding buffer are you using? I am using the binding buffer provided with Kit of Invitrogen.
Dear Elizabeth Gordon
I cant work for specific transcription factor as I have just cloned promoter and I have to isolate transcription factors that bind with it.
How much small size of a probe can I use? One thing that can be done is to find the putative TF binding site, and then design the probe containing that particular binding site so that a specific TF can bind with it. Is it o.k?
and one thing me uisng Molecular Probe kit which utilizes the use of SYBR Green and SYPRO Ruby and with these I cant use nonspecific competitor.
My EMSA gel result is attached here along with key of how much DNA or protein I have used. Kindly check and suggest.
Dr.ALDOORI,,, Looking at your gel,, the ladder bands are not very distinctive ones,, I believe you need to look to parameters of buffering ,i.e/pH,temp...etc.
No.2/how pure are your protein samples ,,when we did the running in our lab/some years ago (HA HA),, there were those minicon cells to get neat samples.
Kindly tell me your other experiences with SYBR Green &SYPRO Rubi EMS.
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