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biological reagents
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misc methods

> The pRSET System

> Purification of His-Tag Proteins

> Protocol for BID Protein Purification

Protocol for BID Protein Purification

  1. 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 30 C until it reached an OD600 of 0.4 - 0.5
  2. at this point the cells were induced with 0.3M NaCl and incubated for 4 more hours before harvesting by centrifugation
  3. the cells were resuspended in 30 ml of lysis buffer
    Lysis Buffer
    13% sucrose
    50 mM NaPO4 pH 8
    1% Triton
  4. lysozyme was added to a final concentration of 0.25mg/ml
  5. PMSF was added to a final concentration of 1mM and the cells were incubated on ice for 30 minutes.
  6. added PEI to a final concentration of 0.15% and incubate 10 more minutes on ice.
  7. the resulting lysate was centrifuged at 10000rpm for 15 minutes to pellet cellular debris
  8. 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
  9. the purified protein was characterized by SDS-polyacrilamide gel electrophoresis, followed by Coomassie staining
  10. the concentration was determined by Bradford assays