I have been expressing FixAB protein(mutant) in E.coli (Nico21(DE3)). I see my protein (in SDS)is getting expressed but most of the protein is present in Flow through. I still have a little amount left in the elute (100 mM imidazole).
For the large scale I incubate my protein (cell lysate)with the resin overnight by stiring I feel like this is not helping.
I need a better solution for this.
Thank you
There are a few other reasons why your protein may not bind to the column: It may be aggregated (but soluble), or the tag may not be sufficiently exposed. You can try moving the tag from one end of the expression construct to the other.
Cobalt ions should be used after evaluating nickel ions and have adsorption with these.
Are you incubating the total cell lysate with your resin? Usually, I lyse the cells and then centrifuge to separate the soluble from insoluble components. Then, you can check on SDS whether your protein is located in your soluble or insoluble fraction. If it is insoluble, it may not bind to your resin.
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