hello,
I am doing EMSA experiments but for sometime, I am getting an additional band of higher size along with the free probe I run but no such band is visible which I amplified and purified the promoter for probe preparation. single band was obtained on amplification by PCR and even after purification by gel elution (Kit). Kindly suggest me the possible mistake I might be doing. Thanks.
is the extra band naked DNA or DNA-protein complex? What if you do not add cell extract?
In case it is a DP complex, did you try competing with increasing excess of unlabelled probe. Do you include Poly-dIdC in your buffer? Also, if your consensus is well defined, try adding a non-binding mismatch probe as competitor. In the past I did many EMSA and some batches of ds-Oligo probes were probably more than the expected dsDNA structure and bound additional 'non-specific' complexes.
Hope this helps!
thank you prof. Stephane.
I am using poly-dIdC in my buffer but the problem I am facing is the negative control which ideally should have only radio labeled DNA is also showing additional band of higher size similar to that of the bound complex. Initially I thought that it might be because of the cross contamination while loading the sample in the gel but was consistent even if no protein was added in any well. I am not able to understand why my probe alone is showing 2 bands even without the addition of protein.
This has happened repeatedly for 2-3 times now.
Please no prof :-) I am just a 'normal' scientist.
my feeling is that your probe is not nice DS DNA but a more complex mix of structures, you could try not/denature it and load both on acrylamide gel to resolve the DS and SS bands. Extracting DNA from acrylamide is possible (see protocols for target definition assay) but quite messy and you will get your hands dirty doing it.
Good luck
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