i am currently working on e.coli IBs purification using Microfiltration system for washing's instead of centrifuge, after solubilisation and refolding unwanted proteins/molecules are getting precipitating between pH 3 to 5, what might be the problem and what are those molecules?
Refolding is probably not 100% efficient, leaving some of the protein in a partially non-native state that precipitates. Or, it could be the refolded protein, if it is unstable at acidic pH.
If the process of removing whatever was used for solubilization is fast, you could be contributing to precipitation.
The causes of precipitation might be unsuitable buffer conditions (pH, salt concentration, additives), high protein concentration, and/or presence of contaminants or misfolded proteins that may trigger precipitation of proteins. As it is mentioned above, the precipitates might contain misfolded proteins or your desired proteins.
Probably oligomers of the protein and/or misfolded protein precipitated. Conduct gel electrophoresis of suspension your precipitate in non reducing condition (sample preparation without DTT or 2_ME) and see MW.
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