Can anyone tell me a rough protein final concentration for a good start?
I am using 20fmol biotinylated DNA in 20ul reaction volume. What can be good amount of DNA binding protein to start with?
20 fmol DNA/20 µl is 1 nM. The lowest concentration of binding protein to use in the titration should therefore be about 10-fold lower, or 0.1 nM, if the binding affinity is very tight, so that you can see the low end of the full dynamic range of binding. The upper end of the concentration range depends on the affinity of the interaction. You should titrate up to at least 10 or 20 x the Kd of the binding interaction to get to near-complete DNA binding.
If you are using nuclear extracts, a range between 5-20 microgram protein would be a good start. If using a recombinant purified protein, 0.1-10 ng may be good to start. Again, it may depend upon the affinity of the protein to bind target DNA sequence.
20 fmol DNA/20 µl is 1 nM. The lowest concentration of binding protein to use in the titration should therefore be about 10-fold lower, or 0.1 nM, if the binding affinity is very tight, so that you can see the low end of the full dynamic range of binding. The upper end of the concentration range depends on the affinity of the interaction. You should titrate up to at least 10 or 20 x the Kd of the binding interaction to get to near-complete DNA binding.
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