Protein isolation - Small size His-tag Protein isolation (Mar/04/2007 )

I was trying to isolate his-tag protein passing thru Ni-NTA column. But protein was not eluted in the elution buffer. Found in wash flow thru and also flow through. Imidazole conc for wash buffer was 5mM and elution buffer 50mM.Could you please help out?

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did you follow the instructions of the manufacturer? a troubl shooting may be provided;

your problem can arise from too much salt especially bivalent cations in your loading solution, or an exhausted Ni-NTA matrix

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How much of the protien do you bind, comapring the flow through with starting material? If you do bind a lot, then you could increase the imidazole conc. to 250mM to elute.

There is always some protein always in washes and flowthrough.

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I am trying to isolate protein in small scale from 25ml culture. Used 2ml column packed with 1ml Ni-NTA. Higher conc of 400mM Imidazole was also taken in elution buffer but it was found in wash and flow through. 300mM NaCl was used in elution buffer, 150mM NaCl in other buffers.

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Is your protein in IB or cytoplasm expressed? Do you desalt your protein against binding buffer before loading? How do you prepare your cell extract before loading on the column? Do you check pressure on the column when you load and wash your sample on column?

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