I have purified my protein-Holliday junction complex by SEC, but the purified complex is not stable. The protein dissociates from the DNA. I would want the complex to be stable to perform further analysis. Any ideas on how to keep them together? Based on EMSA experiments, the affinity between the two is in the nM range though. So, I am wondering what makes them dissociate? I feel they are behaving so in isolation, but when I mix the protein (two oligomeric forms) with the HJ and then see the complex in AUC they are quite stable. So, I believe it is the isolation that drives them crazy. Do you have a better explanation for this and also ways to stabilize the protein-HJ complex ?
EMSAs quite often indicate higher affinities, this is mainly based on the so-called caging effect caused by the gel matrix. MicroScale Thermophoresis (MST) in contrast is a technology to investigate biomolecular interactions free in solution without any immobilization or matrix present. In addition, affinity measurements can be performed in any buffer, even complex bioliquids such as cell lysates or serum. (For more information on the MST technology please visit http://www.nanotemper-technologies.com//home/ or contact me directly via [email protected]).
For many Protein-DNA interactions we could identify stabilizing buffer condiitions at pH7.0 and 50-100mM NaCl.
Depending on the nature of your protein, you might also add non-hydrolysable ATP-analogues (helicases) or divalent cations (nucleases).
Thanks Heide so much. I was also thinking of adding ATP-G-S, but for a different reason, but this seems logical too.
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