I precipitated my his tagged protein by saturated ammonium sulfate and load it on Ni/NTA column without any treatment. after that I do SDS-PAGE and there isn't any band.
If you precipitated the protein you must have dissolved it again for imac. This must have decreased the concentration of ammonium sulfate. Could you describe the whole procedure?
You should centrifuge your ammonium sulfate cut to pellet your target protein.
Then re-suspend the protien pellet in an appropriate loading buffer for IMAC, e.g. 50 mM HEPES pH 7.5 , 500 mM NaCl, 10 mM Imidazole, (choose another suitable buffer if the pI is not compatible).
Centrifuge or filter to remove particles and insoluble material and apply to your column.
It always good practice to retain a small portion at each step when you run SDS-PAGE, it helps identify the step where things go wrong, but also shows how efficient your purification method is.
Adding to what Kou Hayakawa has contributed, I also do suggest that you dialyse you protein after dissolving in appropriate buffers. If high salt remain in your preparation it may affect binding to the column.
Do not think so given the extra dilution due to the chromatography. Your his tag could be hidden. Dissolve your sample in 8 M urea and run imac to corroborate this.
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