Available from Riken BioResource Center.

Plasmid & Protein Purification Database -Purification-

Locus tag ST2172
Plasmid ID 119
Purification Lot ST2172_1e
Purified Date 2004.10.21-12.7
Protein conc. (mg/ml) 15.5
Total volume (ml) 0.15
Total protein (mg) 2.33

Culture Lot:

date host cell scale label weight of bacteira (g)

Cell Lysis and Heat Treatment:

22.8 g of E. coli.
110 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 50mM NaCl.
Sonication, output 8, duty 50/1.5 min, 7 times.
Heat 10 min, 70 deg C.
Centrifuge at 100,000 rcf, 60 min.
170 ml of supernatant.

Purification Procedure:

Step Column Buffer Gradient 1 Gradient 2 FlowRate (ml/min) Eluted
1 Resource Phe 6ml 20mM Tris-HCl(pH8.0),5mM B-ME,1.35M AS 1.35M AS 0M AS 6 805mM AS
2 Resource Q 6 ml 20mM Tris-HCl(pH8.0),5mM B-ME 0M NaCl 2M NaCl 6 146mM NaCl
3 Bio-Scale CHT10I 10mM KPi(pH7.0),5mM B-ME 10mM KPi 500mM KPi 2 36.95mM KPi and 71.25mM KPi
4 Resource S 6 ml 50mM MES(pH6.0),5mM B-ME 0M NaCl 2M NaCl 2 266mM NaCl
5 HiLoad 16/60 Superdex200 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     1 66min


Products were confirmed by MS. At the end of the 1st purification step, two peaks were observed at 805 mM AS and 0 mM AS. Each of them were subjected by an ion exchange column at the 2nd step. At the end of 3rd step, the former peak was divided into two (a and e) and the later peak was divided into three (b, c and d). They were subjected, respectively, to a gel purification column at the next step of purification.