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Plasmid & Protein Purification Database -Purification-

Species Sulfolobus tokodaii
Locus tag ST1241
Products aspartate kinase
Plasmid ID 0071
Vector  
Purification Lot ST1241_1a
Purified Date  
Protein conc. (mg/ml) 2.4
Total volume (ml) 0.625
Total protein (mg) 1.5

Culture Lot:

date host cell scale label weight of bacteira (g)
         

Cell Lysis and Heat Treatment:

25.8 g of E. coli cells.
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110ml of 20mM Tris-HCl(pH8.0), 5mM B-ME, 50mM NaCl.
↓
Sonication, output 8, duty 50/1.5min, 5 times.
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Heat 10 min, 70 deg C.
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Centrifuge, 100,000 rcf, 60 min.
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Supernatant 125 ml.
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Purification Procedure:

Step Column Buffer Gradient 1 Gradient 2 FlowRate (ml/min) Eluted
1 Resource Phe 6 mL 20 mM Tris-HCl (pH8.0), 5 mM b-ME, 1.50 M AS 1.50 M AS 0 M AS 6 0 M AS
2 Resource Q 6 mL 20 mM Tris-HCl (pH8.0), 5 mM b-ME 0M NaCl 2M NaCl 6 192 mM NaCl
3 Bio-Scale CHT2I 10 mM KPi (pH 7.0), 5 mM b-ME 10mM KPi 500mM KPi 1 144 mM KPi
4 Superdex 200 10/300 GL 20 mM Tris-HCl (pH8.0), 5 mM b-ME, 150 mM NaCl     0.5  
5 HiPrep 26/10 Desalting 53ml 20 mM Tris-HCl (pH8.0), 5 mM b-ME        
6            
7            
8            
9            

Comment:

Precipitated pellet at 1.50 M AS was mixed at purification step 2 after desalting.

2011.10.20

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