Calibration for protein-DNA complexes
Hi All,
I am a new MP user, but I am amazed so far. I have a DNA polymerase holoenzyme consisting of a catalytic subunit, a homodimer processivity factor, and a DNA primer-template when fully assembled. I could already see the dynamic distribution between the monomer and dimer running the processivity factor alone. I would like to determine the state of dimerization in complex with DNA and the catalytic subunit. How would you calibrate for that measurement? Thanks in advance!
114 Views
Hi Gabor, thanks for your query!
This is a challenging question; proteins require a protein calibration, nucleic acids need a nucleic acid calibration, and so on. So for complexes you will need to have both - although, even then it may not be perfect as you will have a mixture of both protein / DNA.
You then measure your protein and apply protein calibration. You then measure your DNA and apply DNA ladder calibration (please ensure to use PLL coated slides).
Then you can make a measurement of your complex - look at the shift and apply both calibrations. You may also need to do your complex on both glass and PLL coated glass, depending upon how well the complex adheres to the surface.
There is also the complication factor that if the binding affinity is weak at low concentrations you may also see dissociations. We'd recommend looking at: https://www.refeyn.com/amp/studying-protein-dna-interactions-with-mass-photometry-one-user-s-story to learn more.
Hope this helps!