Ni-NTA QUIAGEN NiNTA purification manual

NiNTA

FREE Online Download Ni-NTA Qiagen NiNTA purification manual pdf

Optimal expression of recombinant proteins in various expression systems including E. colican be easily achieved when the vectors and host cells are carefully chosen, and thegrowth conditions are properly controlled. Culture conditions and the induction of expressionhave profound effects on the way the recombinant protein is produced, and in this contextdirectly influence the strategies employed for protein purification. It is therefore advisableto empirically establish optimal conditions with small-scale cultures before purification ona larger scale is attempted. Recombinant proteins expressed in E. coli can be producedin a soluble form, but in many cases, especially at high expression levels, they aggregateand form insoluble inclusion bodies. The formation of inclusion bodies is influenced by thenature of the protein, by the host cell, and by the level of expression resulting from thevector choice and the growth and induction conditions. Inclusion bodies invariably limitthe utility of standard purification procedures which rely on the protein’s native solubleform. Purification of 6xHis-tagged proteins by Ni-NTA affinity chromatography, however,can be performed under native or denaturing conditions and is not affected by problemsarising from protein insolubility. Most proteins in inclusion bodies are simply solubilizedwith detergents or denaturants such as 8 M urea or 6 M GuHCl before the purificationsteps are initiated.The basic principles pertaining to the Ni-NTA affinity purification procedure under nativeor denaturing conditions are outlined in the flowchart (Figure 20); more details appear inthe following sections.

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