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| Solution Information | help | |
| Enzyme: | MKP-3 | |
| inhibitor: | BDBM46224 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | MKP-3 Assay protocol Materials 1) MKP-3 protein: MKP-3 plasmid was obtained from Prof. John Lazo (University of Pittsburg). MKP-3 recombinant protein was expressed at the Sanford-Burnham Institute Protein Expression and Purification Facility according to the following protocol. Overnight culture of BL21 cells containing pET 21a - 6-His-MKP3 was grown in LB/amp overnight at 37 degrees Celsius; 100ml of the culture was added to 700 mL LB/amp and continued growing at 37 degrees Celsius until OD600 = 0.6-1.0. After induction with 1 mM IPTG, the culture was transferred to 28 degrees Celsius and grown for 3h. Harvested cells were resuspended in 30 mM Tris-HCl, pH 7.9, containing 500 mM NaCl, 2 mM DTT, 10% glycerol (binding buffer) and frozen in liquid nitrogen. Cells were resuspended in 25 mL binding buffer supplemented with 4 mM DTT and the protease inhibitors, and lysed in cell a homogenizer. The supernatant was loaded on a 2-mL Ni-NTA column and, after extensive column washing; MKP-3 was | |
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