Purifying IgG1 from Hybridoma Supernatant

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Material

  • Supernatant (100-200 ml)
  • Peristaltic pump
  • 1 ml HiTrap ProteinG column (binding capacity = 25 ml)
  • PBS (100 ml)
  • 10x PBS
  • .1 M glycine pH 2.7 (25 ml)
  • 1 M Tris pH 9.0 (10 ml)
  • 10 Eppendorf tubes
  • 20% ETOH/H2O (25 ml)

Procedure

  1. Add 0.1 volume 10X PBS to supernatant and filter througn 0.22 µm filter to prevent clogging. Keep until ready to run on column
  2. Pump atleast 5 ml of ddH2O through column at 1 ml per minute
  3. Pump 10 mls PBS through column
  4. Pass supernatant over the column twice. (Save supernatant)
  5. While waiting, prepare 10 labeled tubes for collecting fraction, and put 100 µl of 1M Tris pH 9.0 inside
  6. Wash column with 15 ml PBS, then stop pump
  7. Collect 1 ml fractions into the preprepared tubes by eluting in 0.1 M glycine, pH 2.7. Collect 10 fractions, and mix immediately with Tris to bring pH to 7.0
  8. Wash column with 10 ml PBS
  9. Wash column into 10 ml 20% ETOH and store at 4 C.
  10. Quantitate protein with BioRAD reagent and dialyze into PBS.