| Assay Method Information | |
| | Inhibition LYP ActivityAssay |
| Description: | Expression and Purification of the LYP Catalytic Domain N-terminal (His)6-tagged LYP catalytic domain (residues 1-303) was subcloned into pET28a. For protein expression, the LYP expressing construct was transformed into Escherichia coli BL21-(DE3). Transformed cells were grown at 37° C. in Luria broth (LB) containing 100 μg/mL ampicillin for 4 h until the OD600 reached 0.6 and then induced for growth overnight at room temperature with 0.4 mM IPTG. Cells were harvested by centrifugation (6000 rpm for 15 min at 4° C.), and the cell pellets from 1.5 L of LB medium were suspended in 30 mL of ice-cold lysis buffer consisting of 5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl (pH 7.9), 0.05 mg/mL trypsin inhibitor, and 0.1 mM PMSF. The suspensions were passed twice through a French press at 1000 psi, and the cell lysates were centrifuged at 4° C. for 30 min at 15000 rpm. The supernatants were mixed with 2 mL of Ni-NTA agarose (His*Bind Resin) (Qiagen) at 4° C. for 1 h, and then the mixture was transferred to an empty column. The column was washed with 200 mL of binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl (pH 7.9)), followed by 20 mL of wash buffer (20 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl (pH 7.9)), and then eluted with 20 mL of elution buffer (200 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl (pH 7.9), 5 mM DTT). The elution was dialyzed for 6 h at 4° C. against 1 L buffer A (50 mM NaCl, 20 mM MES (pH 5.8), 1 mM EDTA) and then loaded onto a Mono S column equilibrated at 4° C. with buffer A. The column was washed with 10 mL of buffer A and then eluted with a 40 mL of linear gradient of 0-1 M NaCl in buffer A. The column fractions were analyzed by measuring the absorbance at 280 nm and by carrying out SDS-PAGE analysis. The fractions were combined, concentrated at 4° C. to <1 mL using an Amicon concentrator, and then loaded onto a gel filtration column Superdex 75. The column was eluted with buffer A, and then the fractions which contained protein were combined and concentrated to 8 mg/mL and stored at −80° C. The LYP preparation was shown to be homogeneous by SDS-PAGE analysis. |
| Affinity data for this assay | |
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