Prev.
Available from Riken BioResource Center.

Plasmid & Protein Purification Database -Purification-

Species  
Locus tag APE0092
Products  
Plasmid ID 4003
Vector  
Purification Lot APE0092
Purified Date 2007.3.5-3.13
Protein conc. (mg/ml) 13.3
Total volume (ml) 0.7
Total protein (mg) 9.3

Culture Lot:

date host cell scale label weight of bacteira (g)
        40.1

Cell Lysis and Heat Treatment:

40.1 g of E. coli.
↓
200 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 150mM NaCl.
↓
Sonication output 8, duty 50/4min, 4 times.
↓
Heat 10 min 80 deg C.
↓
Centrifuge at 100,000 rcf for 60 min.
↓
195 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,1.2M AS 1.2M AS 0M AS 2 0.77M AS
2 Resource Q 6 ml 20mM Tris-HCl(pH8.0),5mM B-ME 0M NaCl 0.4M NaCl 5 0.25M NaCl
3 Bio-Scale CHT10I 10mM KPi(pH7.0),5mM B-ME 10mM KPi 500mM KPi 2 Flow through
4 HiLoad 16/60 Superdex 75 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     1 84ml/120ml
5            
6            
7            
8            
9            

Comment:

Products (34 kDa (main), 32 kDa) were confirmed by Trp-MS.

2011.10.20

- kuramitsuxxxx_html.pl