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Plasmid & Protein Purification Database -Purification-

Species  
Locus tag APE0464
Products  
Plasmid ID R4244
Vector  
Purification Lot APE0464
Purified Date 2005.9.13-10.4
Protein conc. (mg/ml) 11.02
Total volume (ml) 1.03
Total protein (mg) 11.35

Culture Lot:

date host cell scale label weight of bacteira (g)
        37

Cell Lysis and Heat Treatment:

21 + 36 g of E. coli.
↓
100 + 200 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 150mM NaCl.
↓
Sonication, output 9, duty 50/2min, 3 times.
↓
Heat 15 min, 80 deg C.
↓
Centrifuge at 100,000 rcf for 50 min.
↓
100 + 120 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,1.05M AS 1.05M AS 0M AS 4.0 0.82M AS
2 Mono Q HR10x10 20mM Tris-HCl(pH8.0),5mM B-ME 0.1M NaCl 0.6M NaCl 2.5 0.32M NaCl(from 0.3M NaCl to 0.5M NaCl)
3 HiLoad 16/60 Superdex 75 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     1 65ml/120ml
4            
5            
6            
7            
8            
9            

Comment:

Three peaks were observed at the Mono Q step and added them together. A bload peak was observed at the initial phase of gel purification step, and producst at the later phase were recovered.

2011.10.20

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