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Available from Riken BioResource Center.

Plasmid & Protein Purification Database -Purification-

Species  
Locus tag APE0392
Products  
Plasmid ID 4015
Vector  
Purification Lot APE0392_1
Purified Date 2005.10.13-10.27
Protein conc. (mg/ml) 0.54
Total volume (ml) 8.8
Total protein (mg) 4.77

Culture Lot:

date host cell scale label weight of bacteira (g)
        56.1

Cell Lysis and Heat Treatment:

56.1 g of E. coli.
↓
130 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 150mM NaCl.
↓
Sonication, output 8, duty 50/2min, 5 times.
↓
Heat 15 min, 80 deg C.
↓
Centrifuge at 100,000 rcf for 60 min.
↓
112 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,1.2M AS 1.2M AS 0M AS 5 0.55M AS
2 Resource Q 6 ml 20mM Tris-HCl(pH8.0),5mM B-ME 0M NaCl 0.6M NaCl 5 0.38M NaCl
3 Bio-Scale CHT5I 10mM KPi(pH7.0),5mM B-ME 10mM KPi 300mM KPi 1 108mM KPi
4 HiLoad 16/60 Superdex200 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     1 60ml/120ml
5            
6            
7            
8            
9            

Comment:

Products (38 kDa) were confirmed by Trp-MS. Further concentration was abandoned because of precipitation.

2011.10.20

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