Prev.
Available from Riken BioResource Center.

Plasmid & Protein Purification Database -Purification-

Species  
Locus tag ST0079
Products  
Plasmid ID 0522
Vector  
Purification Lot ST0079_1a
Purified Date 2005.3.1
Protein conc. (mg/ml) 0.69
Total volume (ml) 8
Total protein (mg) 5.5

Culture Lot:

date host cell scale label weight of bacteira (g)
        34.0

Cell Lysis and Heat Treatment:

34 g of E. coli cell.
140 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 50mM NaCl.
Sonication, output 8, duty 50/2min, 4 times.
Heat 15min, 75 deg C.
Centrifuge at 100,000 rcf, 60 min.
 
 
 
 

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,1.05M AS 1.05M AS 0M AS 2 0M AS
2 Resource Iso 6mL 20mM Tris-HCl(pH8.0),5mM B-ME,1.20M AS 1.20M AS 0M AS 3 900mM AS(between 1.0M AS and 660mM AS)
3 Resource Q 6mL 20mM Tris-HCl(pH8.0),5mM B-ME 0mM NaCl 500mM NaCl 3 240mM NaCl
4 Biocale CHT10I 10mM KPi(pH7.0),5mM B-ME 10mM KPi 500mM KPi 2 between 120mM KPi and 320mM KPi
5 Resouce Q 6mL 20mM Tris-HCl(pH8.0),5mM B-ME 0mM NaCl 500mM NaCl 3 280mM NaCl
6            
7            
8            
9            

Comment:

At the end of Purification 4, solution was divided two groupes, 1a and 1b. Products of the main band (43kDa), 35 kDa and 31 kDa (C terminal half) were confirmed by Trp-Mass.

2011.10.20

- kuramitsuxxxx_html.pl