THANK YOU for replying!
I was getting a bit worried because as usual, everything is on a deadline! Anyway, ~I wanted to answer your questions real quick so you can get a better handle on what i'm doing.
>1) are you using DNases at any step during the EMSA or DNAse inhibitors? these can bind to your DNA sequence and cause shifts on the EMSA
No...I'm not using DNases or DNase inhibitors, however I do use a protease inhibitor cocktail which I add to all my samples prior to incubation/running. It may be this, but
the double probe band does not appear for all my probes, only 2 out of 5 of them, but the two are the key fragments (as usual)
2) your DNA can alter in its running due to changes in salt / buffer / purification and other steps you do after the running of the agarose gel to check it
Following agarose gel running, I perform a normal gel extraction using the qiagen kit, followed by elution in water. Again, all my samples have been treated in the same way, so unless it is a phenomenon tht can happen to some bits of DNA and not others, then I don't think it could be this....hmmm...
3) this is usually considered normal. what is the size of this shift? is it very low on the gel? if it is high, you may have some troubles explaining that as it may approach the range of some of your complexes.
This is where we get to the irritating part - the shift is round about where my complexes are likely to appear....and I believe that they may be masking the complexes
Now I have attached a couple of images for you to see what I have been doing. The first file (EMSA001 s) shows the gel with my positive control DNA to show you the sort of shift I'm expecting (Lanes 1-7). I think the legend on the bottom explains this too. In the same picture, you'll see a promoter region fragment that I know does not have a binding site and therefore does not shift, as expected (Lanes8-10) - the last three lanes are a negative control not expected to bind this DNA at all hence no shift (Lanes 11-13). Good Job so far, right?
The second film I have to show you (EMSA002 s) is of a more dilute range of protein concentrations for my positive control (Lanes 1-7) as I was playing around with protein concentrations to try and find the optimum range. Lanes 8-10 contain the same DNA probe from lanes 8-10 in EMSA001s, which confirms that result. Lanes 11-13 are of another promoter region fragment, this time closer to the start codon of my target gene which run in this double band effect that I'm struggling with. I know that this film is slightly overexposed but I think that the probe level drops ever so slightly across lanes 11-13 showing that there may be binding, but I can't visualise it due to the secondary bands. Incidentally, the DNA probe in Lanes 8-10 is very close in molecular weight to that of the probe in Lanes 11-13, so I believe it is the lower band that I am really interested in?
I'm really sorry for the text overkill in this post, but it's so nice to get a sympathetic, knowledegable ear that I think I went off on one a little bit.
Well there it is...I don't know what to do short of ordering oligos that cover the putative binding site and starting again (not my favourite option)...
What do you think??
I await your comment, but will probably have to post a reply tomorrow as I need to leave the lab for cleaning...I'll try to log on later to check in any case.
Thank you once again for your time...
Canalba, UK EMSA002 s.jpg EMSA001 s.jpg