Whole Cell Project Database -Purification-

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Thermus thermophilus HB8  TTHA1199
ornithine carbamoyltransferase

Lot Information:

Purification Lot: PC011199-42-01-03
sPlasmid ID: PC011199-42
Plasmid Vector: pET-HisTEV
Label: Native
Person: Kanbe I.
Purified Date: 11/16/2006
Total Protein Yield: 7.8 mg
Final Volume: 0.25 mL
Conc.: 31.4 mg/mL

Final SDS-PAGE:

Culture Lot:

date

host cell

scale

SDS-PAGE

11/10/2006Rosetta(DE3)2.5 L

Cell Lysis and Heat Treatment:

8.7g of E. coli cells
Ultra Sonication with 20mM Tris-HCl + 500mM NaCl (pH8.0) + 5mM 2-ME + 1mM PMSF
Heat Treatment at 70°C for 13 min
Ultra Cengrifuge at 200,000G for 1 hr

Purification Procedure:

ColumnBufferFlow Rate (mL/min)Eluted
HisTrap HP 5ml20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 20 mM Imidazole50.5M Imidazole
HiLoad 16/60 Superdex 200 pg20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 20 mM Imidazole1.572.5ml
Protease Treatment  OK
HisTrap HP 5ml20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 20 mM Imidazole5Flowthrough
HiPrep 26/10 Desalting 53ml20 mM Tris-HCl (pH 8.0), 200 mM NaCl7