Originally Posted by sumo1
Well Admin...i wish that was true...but no mixing happened.
I use 1ug of anti-FLAG..and i tried with and without DTT..still no shift...i am thinking n ow..maybe becoz the protein was not binding to the probe in the first place...because the shift was actually from the vector
yes, i did S and NS cold compete...but u see since the vector was binding the probe...it was competed and not competed out respectively with the above mentioned cold competitors.
I still wonder..what could have been IVT/Ted from a empty vector that binds so well to our probe.
is there any other way that i can prove that the shift is from my protein and not the vector??
Ours is a big protein ~100kDa...do u think it will be worthwhile trying in 59:1 bis-acryl ratio...basically trying bigger pore size?? I see some of the probe sticking in the wells...do u think that the protein-DNA complex is not able to come out of the well since the pore size is small (29:1..i use )??
I saw canalban's emsa..pain pics...they were so beautiful...wish mine were too
thanks for replying admin
Thanks for the mention! I have read your post a wee while ago and have been thinking about it. I am also relatively new to the world of EMSAs but I have done a fair bit of digging round for info, plus have got loads of help from this forum - you are in the right place
Several thoughts and things you could try:
1)Could it be that your lysate concentration is too high? Have you done a dilution series across the range? I find that as a range finder, 10 samples of a 1:10 dilution series of the lysate from neat is worth a try.
2)You don't mention whether you carry this out at room temp or in the coldroom. If you are doing this at room temp, then try mixing the DNA and protein in the coldroom and also running the gel at that temperature.
3)The fact that you have some complex being retained in the wells is a bit strange. Could you be precipitating your protein when complex formation occurs? Have a look at your binding buffer components and make sure that the pH and ionic components are ok? I don't know the make up of your binding buffer but I've included mine here for reference.
(25mM HEPES-KOH [pH7.9], 0.1mM EDTA, 5mM MgCl2, 50mM KCl, 1mM DTT, 20% (w/v) glycerol, 0.2mM PMSF, and 1ul protease inhibitor cocktail containing 1.6ug poly (dI-dC) . (dI-dC) (Sigma)
Hope this helps...
Keep posting...we'll get you through this...