Prev.
Available from Riken BioResource Center.

Plasmid & Protein Purification Database -Purification-

Species  
Locus tag APE0674
Products  
Plasmid ID R4254
Vector  
Purification Lot APE0674
Purified Date 2005.8.9-8.19
Protein conc. (mg/ml) 4.45
Total volume (ml) 1.05
Total protein (mg) 4.67

Culture Lot:

date host cell scale label weight of bacteira (g)
        19.4

Cell Lysis and Heat Treatment:

19.4 g of E. coli.
↓
100 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 50mM NaCl.
↓
Sonication, output 8/duty 50, 2 min, 5 times.
↓
Heat 20min, 85 deg C.
↓
Centrifuge at 100,000 rcf for 60min.
↓
 
↓
 
↓
 
 

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 1.2M AS
2 Resource Q 6 ml 20mM Tris-HCl(pH8.0),5mM B-ME 0M NaCl 0.8M NaCl 5 0.35M NaCl
3 HiLoad 16/60 Superdex200 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl 10mM KPi 500mM KPi 2 75ml/120ml
4         1  
5            
6            
7            
8            
9            

Comment:

Products were confirmed by Try-MS.

2011.10.20

- kuramitsuxxxx_html.pl