Huh. I'd never heard of this protocol, but apparently EtBr can in-fact stain protein. Perhaps that's the source of your signal. You could try other stains, such as SYBR Gold or SYBR Green, or using a flourescently labeled DNA (e.g. Cy3).

http://www.ncbi.nlm.nih.gov/pubmed/16597429

Thank you for your reply Dr. Bower. I did in fact read this paper before I posted this. The paper however, says explicitly that proteins will NOT stain with EtBr unless the gel is treated with TCA prior to the staining procedure. Since I did no such treatment to the gel, my question still persists and I am going to try SYBR green in the meanwhile

Hey,

some information would help.

1. Size of your Protein

2. Size and amount of DNA

3. Single or dooubl srand DNA?

4. EMSA Protocol

The Strategy should in fact work but the sensitivity is limited and background can be a problem

Best Sven

Hi Shruti,

Since I don't know how you prepared your protein, I could speculate that there are nucleic acids in your extract. In any case, I agree with Brian that EtBr is not the method of choice. Even without this background your method is not sensitive enough. In lane 9 you can hardly detect your duplex DNA. In case e.g. 5% of your duplex is specifically bound, you have no chance to detect it. In addition, it is not possible to do competition control experiments. If you have no possibility to work with radioactivity, I would try fluorescently labeled oligonucleotides.

I thought of using this kit recently: Lightshift chemiluminescent EMSA kit from Thermo scientific (or active motif). Instead of radio activity, here you are going to biotinylate your DNA and easy detection of the protein DNA complex. (FYI-the company give an offer if we ask for).

In response to your questions Sven, I'm purifying my 11kDa his-tagged protein from an E.Coli whole cell lysate using a Ni-NTA agarose column. I then use an ultra concentrator to get rid of all the imidazole and extra salts and get my protein down to about 2mg/mL. Protein is finally in a HEPES-Tris buffer (50mM HEPES (pH adjusted to 7.5 with 1M Tris-Cl), 150mM NaCl and 5% glycerol).  Protein is theoretically supposed to carry a high positive charge (pI = 9.1).

I have one strand of my duplexes chemically synthesized (single stranded oligos) that I make double stranded by a normal PCR reaction using primers specific for the tail ends of my oligos.

I'm using the reaction buffer composition from Sambrook to set up my reactions i.e. 20ul reactions containing 10% PVA (polyvinyl alcohol), 100-200ug of protein and either 100ng of gel purified duplex or 5uL of the PCR reaction mix [latter was used in the gel picture attached]. I've tried both incubations on ice as well as at room temperature with incubation times ranging from 20-60 minutes.

while incubation goes on, I pre-run my gel in the cold room at 50-80V (I've tried both TBE and tris-glycine buffer systems; all buffers are chilled overnight). I then load the gel and continue running it at 50V for about 6hours.

After the run, I remove the gel, wash it briefly with water and then stain it with an excess of EtBr in 1X TBE for about 15 minutes at room temperature and then image the gel.

Thanks for your insight Dr. Borgmeyer! I guess I'll try increasing my DNA concentration.

I'll also try SYBR green as Dr. Bower suggested

Hi Shruti,

I d strongly recommend using labeled DNA probe (radiolabeled, biotinylated or fluorescently labeled). The sensitivity (and specificity) would be much higher (we are using Bi-labeled probes and using "Chemiluminiscent Nucleic Acid Detection Module"  from Thermo we can easily detect 2 fmoles of the probe.

I am not sure, why are you running EMSA, however generally you run this to check for specific DNA-protein interaction. To be sure, the interaction is specific, it would be good to have a nonspecific competitor in each reaction (poly dIdC, fragmented DNA, ...). In this case, EtBr or SYBR would stain this competitor too, forming terrible smears only. 

Good luck. 

A rapid and sensitive method for detection of proteins in polyacrylamide SDS gels: Staining with ethidium bromide Vincent, A. & Scherrer, K. Mol Biol Rep (1979) 5: 209. https://doi.org/10.1007/BF00782890

• DOIhttps://doi.org/10.1007/BF00782890