Hi friends,
Recently working on some DNA-binding proteins. The dissociation constant is in nanomolar range. Anyone having experience with these DNA binding proteins knows any easy way to characterize the binding activity? Will the interaction be strong enough to survive in PAGE gel?
Thanks a lot!
How about Gel shift assay?
Look at the literature in the following link
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/gel-shift-assays-emsa.html
I think you can keep DNA concentration fixed (obviously in nM range) and then mix with protein at various concentration (go from 1/10 X DNA to 10X DNA. When you run the mix on Agarose gel, you should be able to see the shift in DNA on the gel.
You can estimate Kd from the DNA shift in each case, i.e, the concentration of protein at which DNA mobility was retarded 50%.
It seems from your question that you have already measured the DNA affinity. What method did you use? Are you looking for another method of measuring affinity? Other characteristics of DNA binding proteins you might wish to study include the number and location of nucleotides that are recognized and sequence specificity.
Ramesh, thanks for the advice, I am learning about it..
Adam, actually it is a protein in literature whose DNA affinity has been studied, and the binding sequence is also very clear. I am doing this just to prove that I have got the right protein in its native state, and then I can continue my downstream experiments.
One nice way to measure the affinity of a protein for an oligonucleotide is to use fluorescence polarization. The oligo is synthesized with a fluorescent dye, such as fluorescein, attached to one end. To a fixed concentration of fluorescent nucleotide, a range of concentrations of protein is added. When the oligo binds to the protein, the fluorescence polarization increases. The equipment required is a spectrofluorometer or fluorescence plate reader with polarization capability.
if you have both, the corelating DNA and the protein, you could as well try a dot blot.
Spot the DNA in various concentrations on a nitrocellulose membrane, block it with bsa or milk and use your protein as an "antybody". Then use an antibody against youre protein to detect it by western blot.
This worked very nicely for me, detecting sugar and lipid binding proteins.
On the same principle you could try an ELISA, this way, you are maybe even able to quantify the binding!
A very elegant way to characterize protein-DNA interactions would be MST (MicroScale Thermophoresis). It's an easy, fast and precise method to quantify interactions of any biomolecules directly in solution without any immobilization. You can also choose any buffer to measure the interaction, thus you could nicely mimick conditions as given in the literature you mentioned.
Following the link below you will find lots of examples of protein-DNA interactions analyzed by MST.
http://www.nanotemper-technologies.com/get-it-all/publications/select_category/nucleic-acids/
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