Available from Riken BioResource Center.

Plasmid & Protein Purification Database -Purification-

Locus tag ST0242
Plasmid ID 0153
Purification Lot ST0242_1a
Purified Date 2004.3.23
Protein conc. (mg/ml) 9.5
Total volume (ml) 0.8
Total protein (mg) 7.6

Culture Lot:

date host cell scale label weight of bacteira (g)

Cell Lysis and Heat Treatment:

14 g of E. coli cell.
86 ml of lysate.
Sonication, 0.5 sec on, 0.5 sec off/2min, 8 times.
Heat 15min, 75 deg C.
Centrifuge at 100,000 rcf, 60 min.
80 ml of supernatant.

Purification Procedure:

Step Column Buffer Gradient 1 Gradient 2 FlowRate (ml/min) Eluted
1 Resource Phe 6mL 20mM Tris-HCl(pH7.8),5mM B-ME,1.35M AS 1.35M AS 0M AS 5 500mM AS
2 Resource Q 20mM Tris-HCl(pH7.8),5mM B-ME 0M NaCl 1M NaCl 6 400mM NaCl
3 BioAssist Q 10X10 20mM Tris-HCl(pH7.8),5mM B-ME 0M NaCl 1M NaCl 3 400mM NaCl
4 Bio-Scale CHT 10I 10mM KPi(pH7.0),5mM B-ME 10mM KPi 500mM KPi 3 80mM KPi
5 Bioassist S 10X10 20mM MES(pH5.8),5mM B-ME 0M NaCl 400mM NaCl 2 10mM NaCl
6 HiLoad 16/60 Superdex75 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     0.9  
7 HiTrap heparin HP 20mM MES(pH5.8),5mM B-ME 0M NaCl 500mM NaCl 2.5 100mM NaCl
8 :HiPrep 26/10 Desalting 53ml 20mM Tris-HCl(pH7.8),5mM B-ME        


Supernatant was divided into two, 1a and 1b, and then subjected to further purification.