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Available from Riken BioResource Center.

Plasmid & Protein Purification Database -Purification-

Species  
Locus tag ST2159
Products  
Plasmid ID R0475
Vector  
Purification Lot ST2159
Purified Date 2006.11.7-11.16
Protein conc. (mg/ml) 14.51
Total volume (ml) 0.93
Total protein (mg) 13.49

Culture Lot:

date host cell scale label weight of bacteira (g)
        40.2

Cell Lysis and Heat Treatment:

40.2 g of E. coli.
200 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 50mM NaCl.
Sonication, output 8/duty 50, 3 min, 10 times.
Heat 15min, 75 deg C.
Centrifuge at 100,000 rcf for 50min.
95 ml of supernatant.
 
 
 

Purification Procedure:

Step Column Buffer Gradient 1 Gradient 2 FlowRate (ml/min) Eluted
1 Resource Iso 6ml 20mM Tris-HCl(pH8.0),5mM B-ME,1.2M AS 1.2M AS 0M AS 4 1.0 M AS
2 Resource Q 6 ml 20mM Tris-HCl(pH8.0),5mM B-ME 0M NaCl 0.6M NaCl 5 0.17M NaCl
3 Bio-Scale CHT5I 10mM KPi(pH7.0),5mM B-ME 10mM KPi 300mM KPi 2 53mM KPi
4 HiLoad 16/60 Superdex 75 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     1 75ml/120ml
5            
6            
7            
8            
9            

Comment:

Products were confirmed by Trp-Mass. Products in the sub band arround 50 kDa were also confirmed by Trp-Mass. Size of the products in sub band were reduced after heat long time.

2011.10.20

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