Purification of Immunoglobulin G

Sarah M. Andrew1, Julie A. Titus2

1 Chester College, Chester, 2 National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 16.3
DOI:  10.1002/0471143030.cb1603s05
Online Posting Date:  May, 2001
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Abstract

While unpurified antibodies are suitable for a number of applications, purified antibodies are required for assays based on known concentration of antibody, for chemical modifications such as radiolabeling or conjugation with fluorochromes, or for structural modifications such as production of F(ab')2 or monvalent Fab fragments. This unit contains protocols for purification of IgG by ammonium sulfate precipitation coupled with size‐exclusion chromatography, Protein A‐ and Protein G‐affinity chromatography, immunoaffinity chromatography, and ion‐exchange chromatography.

     
 
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Table of Contents

  • Basic Protocol 1: Ammonium Sulfate Precipitation and Size‐Exclusion Chromatography
  • Basic Protocol 2: Affinity Chromatography Using Protein A–Sepharose
  • Alternate Protocol 1: Affinity Chromatography Using Protein G–Sepharose
  • Alternate Protocol 2: Affinity Chromatography Using Anti–Rat κ Chain Monoclonal Antibody Coupled to Sepharose
  • Basic Protocol 3: DE52 Ion‐Exchange Chromatography with Tris⋅Cl
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Ammonium Sulfate Precipitation and Size‐Exclusion Chromatography

  Materials
  • Ascites fluid or MAb supernatant
  • PBS (see recipe)
  • Saturated ammonium sulfate (SAS; appendix 2A)
  • Borate‐buffered saline (optional; see recipe)
  • Sephacryl S‐200 Superfine (Amersham Pharmacia Biotech)
  • PBS containing 0.02% sodium azide (optional)
  • Glass wool (Polysciences)
  • Sorvall centrifuge and SS‐34 rotor (or equivalent)
  • 26 × 900–mm column (Amersham Pharmacia Biotech)
  • Additional reagents and equipment for protein dialysis ( appendix 3C), column chromatography ( appendix 3A), concentrating proteins ( appendix 3C), and reducing and nonreducing PAGE (unit 6.1 & appendix 3A)

Basic Protocol 2: Affinity Chromatography Using Protein A–Sepharose

  Materials
  • Ascites fluid or MAb supernatant
  • PBS, pH 8.0 and pH 7.3 (see recipe)
  • 1 M NaOH
  • Protein A–Sepharose CL‐4B, hydrated (Amersham Pharmacia Biotech or Sigma)
  • 0.1 M citric acid at pH appropriate for subclass of antibody (see step )
  • Borate‐buffered saline (optional; see recipe)
  • 3 M potassium thiocyanate, filtered
  • Sorvall centrifuge and SS‐34 rotor (or equivalent)
  • 0.45‐µm filter
  • 1.5 × 10–cm column
  • HiTrap protein A column (Amersham Pharmacia Biotech or Sigma; optional)
  • Additional reagents and equipment for dialysis ( appendix 3C) and column chromatography ( appendix 3A)

Alternate Protocol 1: Affinity Chromatography Using Protein G–Sepharose

  • 0.1 M sodium acetate, pH 5.0
  • 0.1 M glycine⋅HCl, pH 2.8
  • HiTrap protein G column (Amersham Pharmacia Biotech or Sigma)

Alternate Protocol 2: Affinity Chromatography Using Anti–Rat κ Chain Monoclonal Antibody Coupled to Sepharose

  • Mouse anti‐rat κ MAb: MAR 18.5 (ATCC TIB 216) purified using protein A–Sepharose (see protocol 2)
  • CNBr–Sepharose CL‐4B (Amersham Pharmacia Biotech)
  • Binding buffer: 0.05 M Tris⋅Cl/0.15 M NaCl/0.02% (w/v) NaN 3, pH 8.6
  • Crude rat antibody solution to be purified (MAb supernatant or ascites fluid)
  • pH 7.0 elution buffer: 0.05 M sodium phosphate/0.15 M NaCl/0.02% (w/v) NaN 3, pH 7.0
  • pH 5.5 elution buffer: 0.05 M sodium citrate/0.15 M NaCl/0.02% (w/v) NaN 3, pH 5.5
  • pH 4.3 elution buffer: 0.5 M sodium acetate/0.15 M NaCl/0.02% (w/v) NaN 3, pH 4.3
  • pH 2.3 elution buffer: 0.5 M glycine/0.15 M NaCl/0.02% (w/v) NaN 3, pH 2.3
  • Additional reagents and equipment for preparation of antibody‐Sepharose ( appendix 3A)

Basic Protocol 3: DE52 Ion‐Exchange Chromatography with Tris⋅Cl

  Materials
  • DE52 powder (Whatman)
  • 0.01 M Tris⋅Cl, pH 8.6 ( appendix 2A)
  • 0.5 M NaCl/0.01 M Tris⋅Cl, pH 8.6 (see recipe)
  • Antibody sample (ascites fluid, tissue culture supernatant, immune serum, or ammonium sulfate precipitate)
  • 1.5 × 50–cm column
  • Additional reagents and equipment for column chromatography ( appendix 3A), and ELISA ( appendix 3A) or SDS‐PAGE (unit 6.1)
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Figures

Videos

Literature Cited

Literature Cited
   Akerstrom, B. and Bjorck, L. 1986. A physiochemical study of protein G molecule with unique immunoglobulin G–binding properties. J. Biol. Chem. 261:10240‐10247.
   Cooper, H.M. and Paterson, Y. 1993. Determination of the specific antibody titer. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp.11.16.1‐11.16.13. John Wiley & Sons, New York.
Key References
   Hardy, R.R. 1986. Purification and characterization of monoclonal antibodies. In Handbook of Experimental Immunology, Vol. 1: Immunochemistry D.M. Weir, ed.) pp.13.1‐13.13. Blackwell Scientific, Oxford.
  An excellent and detailed review of methods for purifying antibodies that includes an extensive list of current literature.
   Harlow, E. and Lane, D. (ed.) 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
  The complete text on antibodies; everything you want to know and more.
   A Technical Guide to Antibody/Protein Purification. 1995. Pierce, Rockford, Ill.
  A highly informative supplement to the Pierce antibody‐purification kits.
   Monoclonal Antibody Purification Handbook. 1994. Pharmacia Biotech, Piscataway, N.J.
  This handbook contains the computer program MAb Assistant.
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