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Antibody Purification Protocol
The University of Chicago





Antibody Purification Protocol

Antibody purification is possible because the cepharose beads are conjugated with protein which has an affinity under certain conditions to immunoglobulins. Following, you'll find a basic protocol for antibody purification either by Protein A or Protein G. The purification steps for A or G are the same, however the buffers differ. 1mL of protein A will bind 20mg of antibody and 1mL of protein G will bind 11mg. The facility does offer antibody purification as a service

Protein A Buffers

  • 1M Tris pH 8.0
  • 20mM Tris pH 8.0
  • 0.1M glycine pH 2.5
  • 20mM glycine pH 5.0
  • 1x PBS
  • 0.1M Sodium Acetate/.5M NaCl

Protein G Buffers

  • 1M Tris pH8.0
  • 0.1M Sodium Acetate pH 5.0
  • 0.1M glycine pH 2.5
  • 0.1M Sodium Acetate/.5M NaCl


  • Load protein A or G slurry into a clean column
  • Sterilize with 50% Ethanol
  • Wash Column with 50mL Sterile PBS
  • Adjust the culture supernatent to pH 5.0 for Protein G and pH 8.0 for Protein A
  • Equilibrate column with 10 column Volumes of 0.1M sodium acetate pH 5.0 (protein G) or 20mM Tris pH 8.0 (protein A)
  • Load the culture supernatent and save flowthrough
  • Wash column with 6 column volumes of 20mM Tris pH 8.0 (Protein A only)
  • Wash column with 10 column volumes of 0.1M Sodium Acetate (protein G) or 20mM glycine pH 5.0 (protein A)
  • Add 100uL of 1M Tris pH 8.0 to 10-15 labelled polypropylene tubes for elution
  • Elute antibody off the column with 3-5 column volumes of 0.1M glycine pH 2.5 (both Protein A and G) at a rate of 1mL/tube
  • Strip any excess antibody from the column by running 10column volumes of 0.1M Sodium Acetate/0.5M NaCl stripping buffer.
  • Make a 1:10 dilution of the eluted product (50uL sample + 450uL 1xPBS)
  • Read samples on a spectrophotometer at 280nm against a 1xPBS blank.
  • The conversion from O.D. to mg/mL is (O.D. X Dilution Factor)/1.37