Hello, I am purifying two proteins separately, but want to isolate them as a complex only. The best way I could think was to have them sit on ice together after purification for an extended period of time and then running gel filtration and isolating the peak at the correct MW. Is that the most feasible? Also what keeps them from dissociating later?
Assuming that the two proteins are capable of assembling into a complex in this manner, the method should work if the affinity of the two proteins for each other is high enough relative to the concentrations of the proteins. That is what would allow you to isolate the complex through gel filtration. The complex concentration should exceed the dissociation constant of the complex by a considerable margin (let's say at least 10-fold, but preferably more) because there will be dilution during the gel filtration chromatography. Subsequently, any other investigations of the isolated complex would also have to keep its concentration high enough to minimize dissociation of the partners.
You also have to think about the stoichiometry of the subunits in the complex when planning the mixture step. The calculations should be done using molarity.
Adam B Shapiro is right.
I would add you may need to pay attention to cofactors during in vitro handling. Especially if your targets include enzymes, protein interactions may require cations (Ca, Mg, eg), nucleotides (ADP, CAMP, eg). And of course, always pH.
Hi, if they truly form a complex, you should be able to isolate directly the complex using gentle enough conditions.
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