I am working on an acetyltransferase that is highly unstable. Its pI is 6.65, and its molecular weight is around 18 kDa. The protein elutes at 1M imidazole and begins to precipitate immediately after elution. After testing various pH levels and salt concentrations, I have been able to stabilize it in MES buffer at pH 5.5 with 1M salt and 5% glycerol, by immediately diluting it after collection. The highest concentration I achieved was approximately 6.67 mg/mL, which was only possible by adding 50 mM EDTA post-elution. This addition seemed to stabilize the protein, but I am uncertain if this approach is optimal, and replicating the results has been challenging.
However, when I try to concentrate it using a concentrator, its concentration rapidly decreases after buffer exchange. I have tested the flow-through, and the protein is not present there. I have also tried flushing the concentrator membrane with buffer, but there is no protein stuck to the membrane either. Only a negligible amount is precipitated. I am unable to determine what is happening to the protein. My eventual goal is to crystallize the protein.
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