Immunoaffinity Chromatography

Timothy A. Springer1

1 Center for Blood Research, Harvard Medical School, Boston, Massachussets
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.2
DOI:  10.1002/0471142735.im0802s18
Online Posting Date:  May, 2001
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This unit describes the isolation of soluble or membrane‐bound protein antigens from cells or homogenized tissue by immunoaffinity chromatography. The technique involves the elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. To elute the bound antigen from the immunoaffinity matrix, the antibody‐antigen interaction is destabilized by brief exposure to high‐pH or low‐pH buffer. The use of batch purification of antigens is an alternate procedure and results in shorter column loading times. The detergent octyl β‐D‐glucoside can be used instead of Triton X‐100 for elution. Because octyl β‐D‐glucoside has a high critical micelle concentration (CMC), it can be readily removed by dialysis, as described.

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Table of Contents

  • Isolation of Proteins Using Antibodies
  • Basic Protocol 1: Isolation of Soluble or Membrane‐Bound Antigens
  • Alternate Protocol 1: Low‐pH Elution of Antigens
  • Alternate Protocol 2: Batch Purification of Antigens
  • Alternate Protocol 3: Elution in Octyl β‐D‐Glucoside
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: Isolation of Soluble or Membrane‐Bound Antigens

  • Antibody (Ab)‐Sepharose (unit 8.3)
  • Activated, quenched (control) Sepharose, prepared as for Ab‐Sepharose (unit 8.3) but eliminating Ab or substituting irrelevant Ab during coupling
  • Cells or homogenized tissue
  • recipeTris/saline/azide (TSA) solution (see recipe), ice cold
  • recipeLysis buffer (see recipe), ice cold
  • 5% (w/v) sodium deoxycholate (Na‐DOC; filter sterilize and store at room temperature)
  • recipeWash buffer (see recipe)
  • recipeTris/Triton/NaCl buffers, pH 8.0 and 9.0 (see recipe), ice‐cold
  • recipeTriethanolamine solution (see recipe), ice cold
  • 1 M Tris⋅Cl, pH 6.7 ( appendix 2A), ice cold
  • recipeColumn storage solution (see recipe), ice cold
  • Chromatography columns
  • Ultracentrifuge
  • Quick‐seal centrifuge tubes (Beckman)
  • Additional reagents and equipment for column chromatography ( appendix 3A), preparing antibody‐Sepharose (unit 8.3), SDS‐PAGE (unit 8.4), and silver staining (unit 8.9)
NOTE: Carry out all procedures involving antigen in a 4°C cold room or on ice.

Alternate Protocol 1: Low‐pH Elution of Antigens

  • recipeSodium phosphate buffer, pH 6.3 (see recipe)
  • recipeGlycine buffer (see recipe)
  • 1 M Tris⋅Cl, pH 9.0 ( appendix 2A)

Alternate Protocol 2: Batch Purification of Antigens

  • recipeTSA solution (see recipe) containing 1% octyl β‐D‐glucoside
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Literature Cited

Literature Cited
   Doering, T.L., Englund, P.T., and Hart, G.W. 1995. Detection of glycophospholipid anchors on proteins. In Current Protocols in Protein Science (J. Coligan, B.M. Dunn, H.L. Ploegh, D.W. Speicher, and P.T. Wingfield, eds) pp. 12.5.1‐12.5.14. John Wiley & Sons, New York.
   Ey, P.L., Prowse, S.J., and Jenkin, C.R. 1978. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A–Sepharose. Immunochemistry 15:429‐436.
   Helenius, A., McCaslin, D.R., Fries, E., and Tanford, C. 1979. Properties of detergents. Methods Enzymol. 56:734‐749.
   Johnson, P., Williams, A.F., and Woollett, G.R. 1985. Purification of membrane glycoproteins with monoclonal antibody affinity columns. In Hybridoma Technology in the Biosciences and Medicine (T.A. Springer, ed.) pp. 163‐175. Plenum, New York.
   Kürzinger, K. and Springer, T.A. 1982. Purification and structural characterization of LFA‐1, a lymphocyte function‐associated antigen, and Mac‐1, a related macrophage differentiation antigen. J. Biol. Chem. 257:12412‐12418.
   Plunkett, M.L. and Springer, T.A. 1986. Purification and characterization of the lymphocyte function‐associated‐2 (LFA‐2) molecule. J. Immunol. 136:4181‐4187.
   Rivnay, B., Wank, S.A., Poy, G., and Metzger, H. 1982. Phospholipids stabilize the interaction between the alpha and beta subunits of the solubilized receptor for immunoglobulin E. Biochemistry 21:6922‐6927.
   Schroeder, R., London, E., and Brown, D. 1994. Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)‐anchored proteins: GPI‐anchored proteins in liposomes and cells show similar behavior. Proc. Natl. Acad. Sci. U.S.A. 91:12130‐12134.
   Tsuchiya, T. and Saito, S. 1984. Use of n‐octyl‐β‐D‐thioglucoside, a new nonionic detergent, for solubilization and reconstitution of membrane proteins. J. Biochem. 96:1593‐1597.
   Wilchek, M., Miron, T., and Kohn, J. 1984. Affinity chromatography. Methods Enzymol. 104:3‐55.
   Williams, A.F. and Barclay, A.N. 1986. Glycoprotein antigens of the lymphocyte surface and their purification by antibody affinity chromatography. In Immunological Methods in Biomedical Sciences (D.M. Weir, L.A. Herzenberg, C.C. Blackwell, and L.A. Herzenberg, eds.) pp. 22.1‐22.24. Blackwell Scientific, Oxford.
Key References
   Harlow, E. and Lane, D. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  Describes the critical parameters involved in immunoaffinity chromatography.
   Hjelmeland, J.M. and Chrambach, A. 1984. Solubilization of functional membrane proteins. Methods Enzymol. 104:305‐318.
  Describes the mechanism of activation of Sepharose by CNBr and alternative activation procedures, and lists numerous examples of proteins purified by affinity chromatography.
   Johnson et al., 1985. See above.
   Wilcheck et al., 1984. See above.
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