As I mentioned my bands are sharp and perfect but no free oligonucleotides and I repeated it again using a large volume of labelled probe but I got the same result no free oligonucleotides. Why?
Then it sounds like you aren't using excess amounts of probe. Try using less cell lysate in your reactions, since increasing the amount of probe didn't work.
You are most likely running the free probe off the bottom of the gel.
no because i run about 11 samples, and i got free oligonucleotides only for free probe and 50X competitor
Then it sounds like you aren't using excess amounts of probe. Try using less cell lysate in your reactions, since increasing the amount of probe didn't work.
Hi Mohamed,
I think Neville is right by pointing to the excess amount of probe, because you can detect your free and also the unbound probes. If you're using something like the LightShift EMSA kit from pierce (streptavidin-HRP) it's not uncommon that you run below the system's detection limit with your free oligos in the binding events. When I had these issues using biotin-labeled hairpin-forming probes preincubation with DTT helped a lot to get an all in all better detection of oligos. On the other hand you may (if not already done so) switch to radioactively labeled probes - sensitivity of detection is much better here. HTH, Robert
Either two things: 1) not enough probe or 2) ran out from the gel. You can do one thing to check. Just load less and less protein (or lysates, eg. 10 ug, 5 ug and 2.5ug). If you see the decrease of your bands, you have enough probe and free probe ran out of the gel. Otherwise, you did not have enough.
- Badges
- Science topic