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Available from Riken BioResource Center.

Plasmid & Protein Purification Database -Purification-

Species  
Locus tag APE0457
Products  
Plasmid ID 4018
Vector  
Purification Lot APE0457_3
Purified Date 2005.5.24-6.6
Protein conc. (mg/ml) 1.28
Total volume (ml) 0.96
Total protein (mg) 1.23

Culture Lot:

date host cell scale label weight of bacteira (g)
        20.0

Cell Lysis and Heat Treatment:

20.0 g of E. coli.
↓
100 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 150mM NaCl.
↓
Sonication, output 8, duty 50/2min, 5 times.
↓
Heat 10 min, 85-90 deg C. It takes 20 min to 85 deg C.
↓
Centrifuge at 100,000 rcf for 50 min.
↓
92 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,1.5M AS 1.5M AS 0M AS 6 from 1.01M AS to 0.82M AS
2 Resource Q 6 ml 20mM Tris-HCl(pH8.0),5mM B-ME 0M NaCl 0.5M NaCl 6 0.35M NaCl
3 Bio-Scale CHT2I 10mM KPi(pH7.0),5mM B-ME 10mM KPi 250mM KPi 1 206mM KPi
4 Superdex200HR10/30 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     0.5 27min
5            
6            
7            
8            
9            

Comment:

Products were confirmed by Trp-MS.

2011.10.20

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