Expression system:    pGEX-4T-1 with N-terminal GST , E. coli BL21(DE3)

Induction condition:  1 mM IPTG, 30 °C, 6 h, 200 rpm, Luria LB broth

Purification: 

1) Suspend cells collected from 1 L medium in ice-cold 40 mL Buffer-A

2) Disrupt cells by a CF1 cell disruptor (Constant Systems), 20 kPsi, 4 °C

3) Centrifuge at 12000 x g, 4 °C, 30 min

4) Load supernatant to a 10 mL gravity column filled with 2 mL GSTrap resin (pre-equilibrated)

5) Wash with 50 mL Buffer-A, finally elute with 10 mL Buffer-B.

6) Concentrate into 2.5 mL and change buffer to Buffer C using a PD-10 column. 

7) Store at 4 °C for up to 6 months, store at -80 °C for up to 1 year (avoid repeated freeze)

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Buffers: A: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3;

              B: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0. 

              C: 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 20 % Glycerol

Optimal Condition

Temperature: 

pH: 

Metal ion:

Example condition: