5ml of an overnight culture of E. coli BL21-SI containing the plasmid vectors, was used to inoculate 1L LB medium, which was incubated at 30º C until it reached an OD600 of 0.4 - 0.5
at this point the cells were induced with 0.3M NaCl and incubated for 4 more hours before harvesting by centrifugation
the cells were resuspended in 30 ml of lysis buffer
Lysis Buffer
13% sucrose
50 mM NaPO4 pH 8
1% Triton
lysozyme was added to a final concentration of 0.25mg/ml
PMSF was added to a final concentration of 1mM and the cells were incubated on ice for 30 minutes.
added PEI to a final concentration of 0.15% and incubate 10 more minutes on ice.
the resulting lysate was centrifuged at 10000rpm for 15 minutes to pellet cellular debris
2ml Ni-Agarose colums were used for purification
Column Buffers:
Equilibration Buffer (500 ml)
10 ml 0.5 M NaPO4 pH 8
37.5 ml 4M NaCl
500ul 100mM PMSF
Elution Buffer (40 ml)
0.8 ml 0.5M NaPO4 pH 8
3.0 ml 4M NaCl
2.0 ml 4M imidazole
0.2 % CHAPS
10 % glycerol
the purified protein was characterized by SDS-polyacrilamide gel electrophoresis, followed by Coomassie staining
the concentration was determined by Bradford assays