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Available from Riken BioResource Center.

Plasmid & Protein Purification Database -Purification-

Species  
Locus tag ST2345
Products  
Plasmid ID R0270
Vector  
Purification Lot ST2345
Purified Date 2006.1.17-1.25
Protein conc. (mg/ml) 22.96
Total volume (ml) 2.68
Total protein (mg) 61.53

Culture Lot:

date host cell scale label weight of bacteira (g)
        25.2

Cell Lysis and Heat Treatment:

25.2 g of E. coli.
↓
140 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 50mM NaCl.
↓
Sonication, output 8, duty 50/1.5 min, 5 times.
↓
Heat 15 min, 75 deg C.
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Centrifuge at 100,000 rcf, 50 min.
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95 ml of supernatant.
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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,1.75M AS 1.75M AS 0M AS 5 1.4M AS
2 Resource Q 6 ml 20mM Tris-HCl(pH8.0),5mM B-ME 0M NaCl 0.3M NaCl 4 0.14M NaCl
3 Bio-Scale CHT10I 10mM KPi(pH7.0),5mM B-ME 10mM KPi 150mM KPi 3 44mM KPi
4 HiLoad 16/60 Superdex 75 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     1 72mL/120mL
5            
6            
7            
8            
9            

Comment:

Products were confirmed by Trp-MS.

2011.10.20

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