Dear All,
Can we incubate Supernatant and Ni charged beads for binding at room temperature for overnight? Currently, we do not have a fixed temp room in our building. What about keeping Ni charged beads and supernatant for 4 hour at RT before elution and washing.
Dear Abir,
as long as the protein doesn't degrade, I don't see a problem. Ni-NTA Agarose and MagBeads can be stored at room temperature for a sufficiently long time without change or degradation. We even have used our PureCube particles together with standard autoclaving, with the beads working as before.
It depends entirely on the protein you are purifying - if your protein is sufficiently stable, both thermodynamically and protease stability, there is no problem in doing the purification at room temperature.
Most people work at low temperature to minimize damage to their protein-of-interest. As the binding interaction equilibrates faster at higher temperature, you might not need to incubate your beads and supernatant as long if you can work at room temperature.
Hi;
Storing your protein at room tempearature over night cause protein degrdadation and your protein may loose the activity. One hour incubation of your supernatant with beads is enough to get sufficient amount of protein purification.
Rare is the protein that does not degrade at room temperature overnight. In my experience, though, an hour is plenty of time to bind a His tag to Ni beads, especially at RT, since as Annemarie pointed out reactions occur faster at higher temp. If your protein can take rocking gently at RT for an hour without degrading (run a gel and see), then I don't see why not.
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