I've been trying to obtain a gel shift, but my free probe (biotin-labeled probe run in a lane by itself) always yields multiple bands. I generated the probe by ordering 5' biotinylated primers for my sequence of interest. I used these primers to amplify a ~200-bp region of maize DNA. I ran the PCR product on a gel, excised the appropriately sized band, and purified it using a kit.
The 60-bp control DNA provided with the kit (Pierce Lightshift Chemiluminescent EMSA kit) looks perfect, but my probe DNA has bands all over the place. Any ideas as to where I went wrong?
It honestly looks like you did not completely separate away un-reacted primers from your product. The upper bands are likely your ~200 BP product, while the lower looks like primers. I am basing this on the right three lanes containing the 60-bp control.
As for why there are two bands, it could be mis-annealed after the purification. You could try re-annealing by boiling for 5 minutes 95C and slow cooling to room temp.
Hi, yes, you are likely to have free primers in the mix and you would not see these on an agarose gel when you purify your fragment. Primers are usually in such molar excess that they can contaminate everything, even after gel purification. One thing to note for EMSAs, when using larger fragments, run a 4% neutral acrylamide gel and always use 0.5X TBE as the running buffer. You can run the contaminating primers off the gel. Also, do not use too much DNA in your binding reaction, less is more here. We used to use 32-P and no more than about 2000 cpm per binding reaction, which translated into about 0.2-1.0 ng of DNA probe per reaction.
I agree with Robert and Gregory above, you may have some residual primer DNA in your fragment. How long are your oligos? is there a chance they produce some primer dimer? The "laddering" at the bottom looks a bit like you may have some synthesis precursors in the mix, as they share most of the sequence, they will also give products. I recommend you try your PCR with HPLC (or PAGE)-purified oligos, most companies offer that service. Often its unnecessary, but as you are facing this problem here, you may want to give it a shot! If thats the case, you can find out by running a lane with primers alone....
Another thought: have you tried to use what you have for your EMSA, once a protein is bound it will run MUCH higher and the multiple bands in the probe may not even matter. I used to do 32P-end-labelled radioactive EMSAs and often had more than one band in the free oligo-region. usually the shifted bands were still nice and clear.
Generally in my experience EMSA is a relatively robust technique. Likely even with your problem here it will still work.
Hope, this is helpful.
Good luck!
Thomas :-)
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