Hi,
I am doing an electrophoretic mobility shift analysis with the outer membrane protein of a bacteria and DNA molecules that potentially bind it. These DNA molecules are biotinylated and are detected by chemiluminiscence. The strange thing is I am visualizing a reduction in intensity of the band but do not really see a shift. If I increase the protein concentration, I do get a corresponding decrease in intensity, but have never been able to see a discernible shift. If I compete it with a non biotinylated DNA, the intensity remains the same as that similar to the unreacted DNA control. However I really would like to see a band 'shifted up". I have the following questions
1. Is there any precedence for this kind of a behavior to be decided as gel shift?
2. Can I interpret that the DNA molecule specifically binds the outer membrane preparation?
3. What can be done to observe the traditional shift like that involving a transcription factor?
4. Does anyone have similar experience with gel shifts with OMPs?
Thanks
biochem
