Immunoaffinity Chromatography

Timothy A. Springer1

1 Center for Blood Research Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.11
DOI:  10.1002/0471142301.ns0511s05
Online Posting Date:  May, 2001
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This technique involves elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. Antibodies are coupled to Sepharose and the cell lysate is passed over the column. After washing, the specific antigen is eluted from the column using one of a number of methods: brief exposure to either high or low pH, or using the detergent octyl beta‐D‐glucoside (easily removable by dialysis). All these methods are described in this unit along with a procedure for covalently linking an antibody to Sepharose using the cyanogen bromide activation method.

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Table of Contents

  • 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
  • Support Protocol 1: Preparation of Antibody‐Sepharose
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: Isolation of Soluble or Membrane‐Bound Antigens

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

Alternate Protocol 1: Low‐pH Elution of Antigens

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

Alternate Protocol 2: Batch Purification of Antigens

  • TSA solution (see recipe) containing 1% octyl β‐D‐glucoside

Alternate Protocol 3: Elution in Octyl β‐D‐Glucoside

  • 1 to 30 mg/ml antigen‐specific monoclonal or polyclonal antibody
  • 0.1 M NaHCO 3/0.5 M NaCl
  • Sepharose CL‐4B (or Sepharose CL‐2B for high‐molecular‐weight antigens; Pharmacia Biotech)
  • 2 M Na 2CO 3
  • Cyanogen bromide (CNBr)/acetonitrile (see recipe)
  • 1 mM and 0.1 mM HCl, ice‐cold
  • 0.05 M glycine (or ethanolamine), pH 8.0
  • Tris/saline/azide (TSA) solution (see recipe)
  • Dialysis tubing (MWCO >10,000)
  • Ultracentrifuge
  • Whatman no. 1 filter paper
  • Buchner funnel
  • Erlenmeyer filtration flask
  • Water aspirator
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Literature Cited

Literature Cited
   Cuatrecasas, P. 1970. Protein purification by affinity chromatography. J. Biol. Chem. 245:3059‐3065.
   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.
   Gelb, W.G. 1973. Affinity chromatography: For separation of biological materials. Am. Lab. 81:61‐67.
   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.
   March, S.C., Parikh, I., and Cuatrecasas, P. 1974. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal. Biochem. 60:149‐152.
   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. 1995. Overview of conventional chromatography. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, H.L. Ploegh, D.W. Speicher, and P.T. Wingfield) pp. 8.1.1‐8.1.9. John Wiley & Sons, New York.
   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.
   Wilchek, et al., 1984. See above.
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