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Plasmid & Protein Purification Database -Purification-

Species  
Locus tag ST0242
Products  
Plasmid ID 0153
Vector  
Purification Lot ST0242_1b
Purified Date 2004.3.23
Protein conc. (mg/ml) 11.4
Total volume (ml) 1.95
Total protein (mg) 22

Culture Lot:

date host cell scale label weight of bacteira (g)
        14

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 HiTrap Heparin HP 20mM MES(pH5.8),5mM B-ME 0M NaCl 500mM NaCl 2.5 100mM NaCl
5 HiPrep 26/10 Desalting 53ml 20mM Tris-HCl(pH7.8),5mM B-ME        
6            
7            
8            
9            

Comment:

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

2011.10.20

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