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Plasmid & Protein Purification Database -Purification-

Species  
Locus tag ST1970
Products  
Plasmid ID 0249
Vector  
Purification Lot ST1970_1b
Purified Date 2005.1.4-1.19
Protein conc. (mg/ml) 0.17
Total volume (ml) 1.35
Total protein (mg) 0.23

Culture Lot:

date host cell scale label weight of bacteira (g)
        37.9

Cell Lysis and Heat Treatment:

37.9 g of E. coli.
↓
130 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 50mM NaCl.
↓
Sonication, output 8, duty 50/2 min, 5 times.
↓
Heat 20 min, 80 deg C.
↓
Centrifuge at 100,000 rcf, 60 min.
↓
120 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,1.05M AS 1.05M AS 0M AS 6 Flow through
2 Resource S 6 ml 50mM MES(pH6.0),5mM B-ME 0M NaCl 2M NaCl 6 Flow through
3 Bio-Scale CHT10I 10mM KPi(pH7.0),5mM B-ME 10mM KPi 500mM KPi 2 135mM KPi
4 Resource Q 6 ml 20mM Tris-HCl(pH8.6),5mM B-ME 0M NaCl 2M NaCl 6 112mM NaCl
5 HiLoad 16/60 Superdex 75 20mM Tris-HCl(pH8.6),5mM B-ME,150mM NaCl     1 60min
6            
7            
8            
9            

Comment:

Products were confirmed by MS.

2011.10.20

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