Editing Split Intein Protein Purification (section)

== Protein Purification using ammonium sulphate precipitation<br>  ==

target protein fused to IC (no his tag) using vector&nbsp; 1743 pIC, such as 1741 pIC-sfGFP, can be purified using the following method:<br> 

<br>1. Mix clarified cell lysates of&nbsp; 8H-ELK-IN&nbsp; (1742 p8H-ELK-IN) and pIC-POI (protein of interest, target protein), such as&nbsp;1741 pIC-sfGFP,&nbsp; incubate at room temperature for 10 min to allow association of IC-POI and 8H-ELK-IN.<br> <br>2. Add solid or saturated ammonium sulfate solution (4.32M) to 0.4 M final concentration to the mixture to induce precipitation of the ELK complex. Incubate on ice for a few minutes (~10 min). Spin for 10 min at max speed (microcentrifuge) or 12000 rpm. The pellet contains the target protein noncovalently linked to ELK. 

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3. Resuspend pellet in adequate amount of Buffer A (0.5 M NaCl, 50 mM Tris-HCl, pH 8.0) 

<br>4. Add 1 M or 2 M DTT to final concentratio of 50 mM, incubate at room temperature for 3 or 20 h. 

As low as 1 mM DTT concentration can be used, but reaction time and concentration should be optimized 

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5. Add solid or saturated ammonium sulfate solution (4.32M) to final concentration of 0.4 M to the mixture, incubate on ice for 10 min. Spin at max speed to precipitate the&nbsp; 8H-ELK-IN ~IC complex. 

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6. Remove precipitant by centrifugation. 

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7. The supernatant contains purified target protein 

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