[RASMB] Dissociation constants for protein/DNA complexes

Sarah Kessans sarah.kessans at canterbury.ac.nz
Wed Jan 14 18:20:52 PST 2015


Hi all,

We are trying a new (?) method of determining dissociation constants for protein/DNA complexes using velocity sedimentation and I am looking for feedback regarding the method.

Our method of determining Kds uses fluorescently-labeled oligos in complex with unlabeled protein.  By scanning at 488 nm, we can determine the size distribution of both unbound DNA and the protein/DNA complex.  We then calculate the area under each of the (baseline resolved) distribution curves, normalize these values to 100% total, and use the ratios (along with our known initial concentrations) to calculate the molar concentrations of bound/unbound protein/DNA.  From these data, we are able to get Kds that correspond well to Kds calculated from other experiments.

We are looking to publish these results, and I was wondering if anyone knew of Kds calculated in this manner before (either in published literature or otherwise)?  Any suggestions/comments welcome, thanks!

Best,
Sarah

___________________________
Sarah Kessans
Post-doctoral Fellow
Biomolecular Interaction Centre
School of Biological Sciences
University of Canterbury

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