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Plasmid & Protein Purification Database -Purification-

Species  
Locus tag STS042
Products  
Plasmid ID R0155
Vector  
Purification Lot STS042_2b
Purified Date 2004.8.4-8.24
Protein conc. (mg/ml) 9.92
Total volume (ml) 0.25
Total protein (mg) 2.48

Culture Lot:

date host cell scale label weight of bacteira (g)
        20.5

Cell Lysis and Heat Treatment:

20.5 g of E. coli.
105 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 150mM NaCl.
Sonication, output 8/duty 50, 2 min, 3 times.
Heat 15min, 75 deg C.
Centrifuge at 100,000 rcf for 60min.
90 ml of supernatant.
 
 
 

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,1.5M AS 1.5M AS 0M AS 6 1.5M AS
2 BioAssist Q 10x10 20mM Tris-HCl(pH8.0),5mM B-ME 0M NaCl 0.5M NaCl 3 0.13M NaCl
3 HiLoad 16/60 Superdex 75 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     1 80ml/120ml
4            
5            
6            
7            
8            
9            

Comment:

Products were confirmed by trp MS. Entire MS comfirmed. Products were devided in the gel filtration step into two peaks, the main peak (a, possibly multimer) and the sub peak (b, possibly monomer). The products in b showed orange. The final buffer contains 150 mM NaCl to avoid absorption to gel and memblane.

2011.10.20

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