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Plasmid & Protein Purification Database -Purification-

Species  
Locus tag ST2341
Products  
Plasmid ID 0269
Vector  
Purification Lot ST2341_2-a
Purified Date 2004.10.6-10.20
Protein conc. (mg/ml) 11.8
Total volume (ml) 1.6
Total protein (mg) 18.9

Culture Lot:

date host cell scale label weight of bacteira (g)
        21.4

Cell Lysis and Heat Treatment:

21.4 g of E. coli.
↓
105 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 50mM NaCl.
↓
Sonication, output 8, duty 50/1.5 min, 5 times.
↓
Heat 10 min, 70 deg C.
↓
Centrifuge at 100,000 rcf, 60 min.
↓
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,1.35M AS 1.35M AS 0M AS 6 1.12M AS
2 Resourse Iso 6ml 50mM MES(pH6.0),5mM B-ME 0M NaCl 2M NaCl 6 64mM NaCl
3 Bio-Scale CHT10I 10mM KPi(pH7.0),5mM B-ME 10mM KPi 500mM KPi 2 79.09mM KPi
4 HiLoad 16/60 Superdex 75 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     1 60min
5            
6            
7            
8            
9            

Comment:

Products were confirmed by MS. There were two peaks at the step of gel purification. The first peak was determined as a and the second was b. Both were determined as the target.

2011.10.20

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