I've run my EMSAs alongside EBNA controls (which are included in the LightShift EMSA kit) and the control lanes migrate fine, therefore I don't think it's an issue with the gel, resolution, transfer or detection. It seems like it has to be an issue with how I prepared the oligo.
Here's how I did that: Following amplification, I purified the oligo from an agarose gel using a GeneClean II kit and I labeled it using Pierce's Biotin 3' End DNA Labeling Kit. I should also note I did not end label the complementary strands separately (as Pierce recommended).
Any ideas?
Some questions arise from your problem :
What is the sequence of your oligonucleotides ? Do they have blunt or cohesive ends ? How di you manage to hybridize them ?
It might be an oligomerization problem due to improper hybridization of your DNA strands.
we need more data
you prepared the oligo by amplification (?)...using primers to PCR? how long is the EMSA probe (if that is what you call oligo)?
Your agarose gel looks pretty good, a single major band with a hint of something smaller which may not be an issue. However, agarose gels will not resolve small fragments very well. So even in that single band, there may be variants. I assume for the EMSA you are running a polyacrylamide gel in 0.5X TBE. For 250 bp probe, a 4% gel is probably best. You will find that this gel resolves fragments so much better that alternative structures may now be seen or even the amount of biotin added could alter the charge to mass ratio.
If you cannot solve this problem, I would suggest isolating your fragment by cutting it out of a 4% neutral acrylamide gel. You can stain this with EtBr just like an agarose gel. This way, only the appropriate double stranded DNA fragment will be used for the EMSA.
The most straightforward way to troubleshoot your problem is as following.
Calculate the concentration of the PCR product after the gel purification and save about 30-40 ng. Modify the rest of your ds DNA probe in the reaction with biotin, purify it and put aside ~30-40 ng of biotinylated purified DNA. Prepare 6% small (~10X10 cm)
0.5X TBE PAAG and run both your DNA samples on it along with the DNA markers that cover for the region of interest (from 100 to 1000 bp). Stop the gel when BpB is about 1 cm from the bottom of the gel. Stain the gel with EtBr for 10-15 min at r.t.. Wash the gel twice with 200 ml of ddH2O and visualize stained DNA products. If you have multiple products either due to the modification procedure or partial DNA degradation (which is unlikely but still...) , you will see that immediately.
Good luck
To be sure that you have purified the product to homogeneity, you can run a small amount of the gel-purified product in a gel (preferably with a wider well, so that the bands are thinner), so then you know if you have contaminants, since the resolution will be better.
Another thing could be some contaminating proteins or carried over proteins (from PCR or Biotin-labelling) that is still bound to some of the DNA, so they run differently.
Good luck.
You could try labeling the oligos with biotin and purify them before using them as PCR primers to generate the probe. Any provider would do a HPLC purification of a biotinylated oligonucleotide and will give you a single band. This approach should solve the problem of heterogenous bands but will reduce your signal since you will have less biotin labels per molecule. If your sensitivity is good then it is no problem.
Good luck.
I am having a similar problem any updated results John?
Please see my post
https://www.researchgate.net/post/What_is_the_band_and_how_can_I_remove_the_non-specific_band_in_my_free_probes?_sg=XL2mQJuj2S5nCUbVg9x1swI1VNnf%2BEqIjFsBnJjPHf5JEk4I09z3qPV18rt8YnzJ
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