I am working on a human helicase protein and when I load them on a SEC column Superdex 200 (GE), I don't get a good recovery. In addition, when I load my protein-DNA complex into the same column the complex does not come out at all. Had anyone experienced a similar problem?
Also, had anyone here tried to purify a protein-Holliday junction complex? I have a mixture of two oligomers (homo) in my protein, and one of the oligomer binds to HJ, while the other does not. For further studies I would like to purify the protein-HJ complex from the non-binding oligomer of the protein, but not finding a good way. Please share your thoughts/experiences.
Thanks for your input. I don't think the protein aggregates, because I have used similar conditions for analytical ultracentrifugation and the protein seems to be happy (w/o aggregation). I am using 10 mM HEPES, pH 7.4, 5 mM MgCl2, 150 mM KCl, 0.5 mM TCEP, 2 % glycerol.
It is strange that the complex does not come out and that the recovery is low. Do you get anything in the void volume, maybe the helicase is not stable and aggregates? How much salt do you use in your buffer for SEC? Did you check the affinity of your helicase for the Holliday junction in the same buffer? Maybe you need to reduce the salt concentration. If the affinity is not high then you will probably not be able to purify the complex like this. Also, what do you mean you have two oligomers, what is the difference between them? What is the difference in MWt between the oligomer that does not bind and the complex?
Hi Diana, Thanks for the response. Please see below my answers/explanations.
I do not see any in the void volume. I also know they do not aggregate, because I have done Sed velocity analytical ultracentrifuge expts, and it clearly shows 1 ) No aggregates and 2) the complex is stable for couple of hours, if not more.
I use 150 mM salt and again, the Sed Vel AUC experiments were done at this salt and it shows the formation of the complex.
The affinity and the activity of my protein in that buffer is good as per our in vitro activity and EMSA assays. The affinity based on gel shift assay is quite high.
The one that binds is twice as big as the one that does not bind.
Thanks !
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