The protein of interest is an intrinsically disordered protein (myosin tail domain) which was denatured to keep it in solution followed by renaturation during dialysation. Finally I can gain the protein in soluble form for experiments but If I do a cleaning UC just before the final utilization a small proportion settle down into pellet. I can use the supernatant as well after that, but with lower concentration.
Does anyone have an idea?
Thank you for your answers in advance!
Some of your protein is still present as high molecular weight aggregates. Although they are not big enough to form a visible precipitate, they will nonetheless pellet upon ultracentrifugation. Most likely, you would also be able to detect them by gel filtration as they would migrate in the void volume.
If the protein is at a high concentration it may form a gel when cooled and this could lead to some degree of insolubility when the temperature is then raised.
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