1. why don't you choose PAGE for EMSA?

2. I think there's no specific requirement for agarose gel except that you should check your DNA size, do not let it flow through.

3. transfer is another issue that you should be carefully handle. it's difficult thant regular PAGE-transfer.

Please revise attached file.

Protocol

Hi Farah! Can I ask you why you are not using native PAGE for your EMSA? I think you are not describing well what you are wishing to do. What do you expect from your EMSA? Is it to detect the binding of a specific protein to your DNA probe? Are you using total protein extract, a purified protein or a protein complex? Are you using double stranded oligos or a PCR probe? How long is your probe? Etc,…

No, we don’t use agarose to separate proteins. It would all have been easy if it was. Just imagine the impact not only for mass spectrometry analysis knowing that agarose can be dissolved using enzymatic or chemical treatment while polyacrylamide cannot.

I forgot to ask you what is your method of detection?

Dear Abdelhalim Boukaba

probe size is 250bp and i am using nuclear protein extract from plant. My aim is to identify transcription factor that bind to promoter region of interest. Promoter sequence is known, I have to identify its binding proteins.

I have tried 6% native PAGE BUT ssample stays in wells except unbound DNA. I have also observed decrease in DNA concentration at bottom with increase in protein concentraion. To move the sample from well, I have tried 4% stacking gel also, but problem did not resolved and sample still in well except unbound DNA.

Detection method that I am using is EMSA kit from invitrogen based on SYBR Green ANd SYPRO RUby.

Then I thought perhapse complex size is large and i need to do EMSA on agarose gel. So I moved to Agarose BUT in Agarose gel, DNA is detected at bottom but when i stained the gel with Sypro Ruby or Commassie, no protein detection. I tried 2ug protein concentration also but no detection. After EMSA I have to proceed that complex to Mass Spectrometry for protein identification.

Kindly guide..

Dear Quan Li

Thanks for protocol.

Hi Farah! First If you are going to submit the bound proteins to Mass spectrometry for identification then I suggest to you this paper which just has just come out (“Unbiased proteomic analysis of proteins interacting with the HIV-1 5'LTR sequence: role of the transcription factor Meis” by Tacheny et al. Nucleic Acids Research (2012) August 16. It is an open access (free to download). Have a look to it because I think it is exactly what you intend to do. It is relatively easy to perform the affinity purification.

I already posted this response to you because I found that you have already tried EMSA with native PAGE. I post it again: I think you should bear in mind that if you are using total cell extract, the fraction of your protein that will bind to the DNA probe is very small and likely you will not be able to see anything meaningful unless you radiolabel your probe or use other non-radioactive methods (DIG, biotin-dUTP, etc…). I am not sure picogreen (which is the fluorescent dye) can detect on gel the fraction of bound DNA fragments.

Also, it is normal if you are using native conditions that a lot of protein may remain in the well, others will migrate in the opposite direction. Remember that in native conditions the migration of the proteins will be determined by their net charge at the pH of the running buffer.

I am also using the flourescent dyes like SYPRO Ruby, and I have seen certain publications of it. Unfortunately in Pakistan, there is not availability of Radioactive assays so I cant use it and same with biotin labbellled. Therefore I have chosen the flourescnet dyes method.

Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions Lance M. Hellman and Michael G. Fried*

I have also used this protocol but no improvement.

Hi Farah! As I told you in your EMSA, it is not an artifact what you are seeing. In native conditions proteins stay in the well, other will migrate toward the anode or the cathode according to their Net charge. That’s what you would expect and changing to agarose will change nothing.

I ‘ve done in the past EMSA with both Native PAGE and agarose gels, but I used radioactive labeling of oligo probes which mean you have to dry the gel. I think, it is quite possible that you have a band shift in your gel but you can’t detect it with Syprogreen dye. Shifting around 500 pg (which is the detection limit of the dye) of a double stranded band is a lot. (you can do the math)

EMSA was not set up for what you intend to do. I really recommend that you use an affinity purification using your DNA probe. You can generate a probe with a 5’-biotin or desthiobiotin label. This is easy because you just have to PCR your probe using a 5’-biotin or desthiobiotin –conjugated oligo, which is readily shipped from the company you order your primers. Just order the same 5’ primer with the modification (they are relatively cheap now). Binding your 5’ biotin or desthiobiotin probe to strepatavidin-coated magnetic beads or strepatvidin-conjugated sepharose or agarose require only an incubation in a buffer. So, follow the procedure in the paper that I recommend you or ask back if you have any kind of questions.. Streptavidin -coated magnetic beads or streptavidin-sepharose gel are not that expensive. You can just incubate your protein extract with the probe bound to the streptavidin sepharose, wash, elute and send for MS protein identification.