Posted 18 October 2010 - 01:30 PM
I am currently running ( or trying to run) EMSA gels. This is my third try and it looks as though the complexes remain in the wells and do not migrate into the gel itself. The free probe however does migrate. I am running the gels at 4 degrees C, in 0.5X TBE at 120 volts for 1 hour and pre-running the gels as well. I have tried increasing the amount of protein, from 5ug to 25 ug, as initially I thought that the lack of bands was due to inadequate amounts of protein. I would appreciate any input anyone could give me.
Posted 19 October 2010 - 05:46 AM
I did a lot of these during my PhD and found that they were a bit fussy.... how big is the protein that you are trying to analyse? I previously worked with an MBP-fusion protein (MBP tethered to an AraC-type protein) and the total size was 80kDa. AraC-type proteins tend to aggregate at high concentrations and so I found that if I applied more than 1ug to the well then most of it would just sit there. I was using a 5% gel by the way - the bigger the protein the lower the polyacrylamide percentage you need (obv)!
One thing you might find useful is to look at the pI of your protein [if you don't know it, there is a program called ProtParam (http://www.expasy.ch.../protparam.html) which will predict it for you from the amino acid sequence]. Since your protein is least soluble at this value, it makes sense to vary the pH of your DNA-binding reaction so that it's not near the pI of your protein - this simple step made my experiments work properly. I also found that inclusion of DTT (a strong reducing agent) in the DNA-binding reaction was necessary to make my protein bind DNA, possibly because it destroys non-biologically relevant disulphide bridges your protein may be forming. This could also make it aggregate and refuse to migrate, so may be worth checking out.
Your other gel conditions sound fine; just be careful to use the same buffer for making the gel and running it! i.e. the same stock of 0.5xTBE is used both in gel preparation and running buffer. Oh, and I would recommend pH'ing your buffers to the same value as used in your DNA-binding reaction. For a general EMSA protocol I used Michel et al 2005 (a paper on PchR) - it's not a methods paper but is very very helpful.
Wow, it's an essay. Good luck!
Posted 19 October 2010 - 06:11 AM
Have a nice day.