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Available from Riken BioResource Center.

Plasmid & Protein Purification Database -Purification-

Species  
Locus tag APE0550
Products  
Plasmid ID R4333
Vector  
Purification Lot APE0550_3
Purified Date 2005.9.15-9.22
Protein conc. (mg/ml) 5.16
Total volume (ml) 0.5
Total protein (mg) 2.58

Culture Lot:

date host cell scale label weight of bacteira (g)
        50.1

Cell Lysis and Heat Treatment:

20.1 g of E. coli.
↓
150.1 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 150mM NaCl.
↓
Sonication, output 8, duty 50/2 min, 8 times.
↓
Heat 10 min, 80 deg C.
↓
Centrifuge at 100,000 rcf for 50 min.
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156 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,1.8M AS 1.8M AS 0M AS 4 1.42M AS
2 Resource S 6 ml 20mM Tris-HCl(pH8.0),5mM B-ME 0M NaCl 1M NaCl 5 0.39M NaCl
3 HiLoad 16/60 Superdex 75 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     1 70min
4 Bio-Scale CHT2I 10mM KPi(pH7.0),5mM B-ME 10mM Kpi 500mM Kpi 1 197.4mM Kpi
5 HiPrep 26/10 Desalting 53ml 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl        
6            
7            
8            
9            

Comment:

Products were confirmed by MS. This sample dended to be degradation. Products were stored with glycelrol.

2011.10.20

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