I have done F- EMSA and used 200nm of my DNA and titrated it with different concentration of my repressor protein. I found the kd to be 1.6 micro molar and I used prism sofware to fit my data with Hills equation for saturation binding.
Is this kd physiologically possible? I guess its too weak affinity for a repressor protein.
I have shown that the binding is happening only in specific DNA and non specific DNA did not show any shift.
We don't do radioactivity in our lab and thats the reason I had started with 200nM of DNA. Is it possible that my DNA concentration is too high and thats the reason why my titration requires uM amounts of protein. Is there any way to interpret the kd for lower amount of DNA with the existing data?
Lac repressor's Kd is in the order of pM so your measured data seem to be a bit off. However, if the cause was using 200 nM DNA, you could not get Kd 1.6 uM because the molecules would tend to bind in 1:1 ratio until saturation (in pretty much linear fashion), giving you a false Kd around 100 nM, assuming the Kd is in the order of pM.
So, it could be that your fluorophore interferes with the binding. You could avoid it by testing londer DNA where the fluorophore is further away from the binding site. But it could be also caused by not having the right conditions - salts (for example Mg2+), some small molecule that is an allosteric activatior of the repressor etc.
And since you are doing fluorescence EMSA and want to avoid radiolabeling, you can also consider using fluorescence anisotropy, you wouldn't have to wait for the electrophoresis.
Good luck!
Dear Jiri Koubek
Thanks for your valuable suggestion. I was wrong by saying Fluorescence EMSA. My DNA was not labelled with any fluorophore. I used sybr Gold to detect my DNA.
And looking my gel it looks like there is more than 1 binding site, because I get atleast three bands. And I have done my EMSA in the presence and absence of Mg2+, and there was no difference.
The kd which I reported is macroscopic.I am trying to get an ITC done tomorrow and update the microscopic kd. (If I get one!)
Thank you a lot!
I think you used too much DNA in your EMSA if you used 200 nmoles. The amount of DNA you should use is on the order of 1-5 fmoles per reaction (for fluorescence) and about 20-30 fmoles per reaction for SYBR-gold. (i.e. use the least amount of DNA that you can possibly detect.)
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