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Available from Riken BioResource Center.

Plasmid & Protein Purification Database -Purification-

Species Aeropyrum pernix
Locus tag APE0056
Products enoyl-CoA hydratase
Plasmid ID R4323
Vector  
Purification Lot APE0056
Purified Date 2005.12.7-12.15
Protein conc. (mg/ml) 8.43
Total volume (ml) 0.76
Total protein (mg) 6.4

Culture Lot:

date host cell scale label weight of bacteira (g)
        38.0

Cell Lysis and Heat Treatment:

Combined lysates of 18.5 and 19.5 g of E. coli.
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100ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 150mM NaCl.
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Sonication output 8, duty 50/4min, 5 times.
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Heat 15min 85 deg C.
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Centrifuge at 100,000 rcf for 50 min.
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98 ml of supernatant.
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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,1.2M AS 1.2M AS 0M AS 6 0.75M AS
2 Resource S 6 ml 20mM Tris-HCl(pH8.0),5mM B-M 0M NaCl 2M NaCl 4 0.14M NaCl
3 HiLoad 16/60 Superdex 75 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     1 64min
4            
5            
6            
7            
8            
9            

Comment:

Products were confirmed by Trp-MS.

2011.10.20

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