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Available from Riken BioResource Center.

Plasmid & Protein Purification Database -Purification-

Species  
Locus tag ST2315
Products  
Plasmid ID 742
Vector  
Purification Lot ST2315
Purified Date 2006.10.3-10.13
Protein conc. (mg/ml) 18.3
Total volume (ml) 3.0
Total protein (mg) 54.9

Culture Lot:

date host cell scale label weight of bacteira (g)
        19.5

Cell Lysis and Heat Treatment:

19.0 g of E. coli.
↓
110 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 50mM NaCl.
↓
Sonication, output 8/duty 50, 5 min, 10 times.
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Heat 15min, 75 deg C.
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Centrifuge at 100,000 rcf for 60min.
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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 5 1.37M AS
2 Resource Q 6 ml 20mM Tris-HCl(pH8.0),5mM B-ME 0M NaCl 0.6M NaCl 4 0.04M NaCl
3 HiLoad 16/60 Superdex 75 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     1 67min
4            
5            
6            
7            
8            
9            

Comment:

Products were confirmed by Trp-MS. However, products showed double band on the electropholesis gel and their mobility differed from expected.

2011.10.20

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