EMSA using Cy3-labelled probe - (Jun/15/2007 )
Please take a look at my EMSA gel. I find it very strange because I don't see any free probes. Does my probe really bind to protein? What has gone wrong?
I think your free probe is likely the blotch at the bottom. I suspect either a physical problem or a technical problem is occurring at some step or another...you may want to troubleshoot a bit and try for a cleaner blot. if it transferred poorly at the edges, that would explain why the 'free probe' band isn't more intense
another thing to consider - we can't troubleshoot well if you don't tell us what you've done
how did the rest of the membrane look? how much probe did you add? what did you see in your control lanes? etc etc....we can't really help you effectively if we don't get anything but a blotchy film at the end....please give us some details of your protocol
This is a native gel using TBE buffer. I want to analyze the binding of my Cy3-labelled DNA probe with my protein using electrophoretic gel shift assay. I used 1ug of probe, 63.92ug of protein of interest and 27.2ug of unprogrammed protein (meaning TNT without any cDNA added for translation). For the binding interaction, I added binding buffer, my protein, poly (dIdC) and my probe. The gel was run in 0.5x TBE.
What can have gone wrong with my results?