Immunoprecipitation

Juan S. Bonifacino1, Esteban C. Dell'Angelica1, Timothy A. Springer2

1 National Institute of Child Health and Human Development, Bethesda, Maryland, 2 Center of Blood Research, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.3
DOI:  10.1002/0471142735.im0803s41
Online Posting Date:  May, 2001
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Abstract

Immunoprecipitation consists of multiple ordered steps: lysing the cell with detergent if the antigen (usually a protein) to be precipitated is membrane‐bound; binding of a specific antigen to an antibody; precipitating the antibody‐antigen complex; washing the precipitate; and dissociating the antigen from the immune complex. The dissociated antigen is then analyzed by electrophoretic methods. In this unit, the basic protocol details the immunoprecipitation of a radiolabeled antigen with a specific antibody (polyclonal or monoclonal) covalently linked to Sepharose. Preparation of Ab‐Sepharose is described in the Support Protocol. The first two alternate protocols present methods for precipitating or isolating the soluble immune complexes formed between a specific antibody and a radiolabeled antigen. Immunoprecipitation is achieved with polyclonal anti‐immunoglobulin (Ig) serum, anti‐Ig‐Sepharose, Staphylococcus protein A or Streptococcus protein G bound to Sepharose, or Staphylococcus aureus bacteria which contain protein A on the cell surface. The third alternate protocol should be used for immunoprecipitation of antigens that are nonspecifically associated with other proteins. The fourth alternate protocol describes immunoprecipitation of unlabeled protein antigens with Ab‐Sepharose.

     
 
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Table of Contents

  • Basic Protocol 1: Immunoprecipitation Using Cells in Suspension Lysed with a Nondenaturing Detergent Solution
  • Alternate Protocol 1: Immunoprecipitation Using Adherent Cells Lysed with a Nondenaturing Detergent Solution
  • Alternate Protocol 2: Immunoprecipitation Using Cells Lysed with Detergent Under Denaturing Conditions
  • Alternate Protocol 3: Immunoprecipitation Using Cells Lysed Without Detergent
  • Alternate Protocol 4: Immunoprecipitation Using Yeast Cells Disrupted with Glass Beads
  • Alternate Protocol 5: Immunoprecipitation with Antibody‐Sepharose
  • Support Protocol 1: Preparation of Antibody‐Sepharose
  • Alternate Protocol 6: Immunoprecipitation of Radiolabeled Antigen with Anti‐Ig Serum
  • Basic Protocol 2: Immunoprecipitation‐Recapture
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Immunoprecipitation Using Cells in Suspension Lysed with a Nondenaturing Detergent Solution

  Materials
  • Unlabeled or labeled cells in suspension
  • PBS ( appendix 2A), ice cold
  • recipeNondenaturing lysis buffer (see recipe), ice cold
  • 50% (v/v) protein A–Sepharose bead (Sigma, Amersham Pharmacia Biotech) slurry in PBS containing 0.1% (w/v) BSA and 0.01% (w/v) sodium azide (NaN 3)
  • Specific polyclonal antibody (antiserum or affinity‐purified immunoglobulin) or monoclonal antibody (ascites, culture supernatant, or purified immunoglobulin)
  • Control antibody of same type as specific antibody (e.g., preimmune serum or purified irrelevant immunoglobulin for specific polyclonal antibody; irrelevant ascites, hybridoma culture supernatant, or purified immunoglobulin for specific monoclonal antibody; see )
  • 10% (w/v) BSA
  • recipeWash buffer (see recipe), ice cold
  • Microcentrifuge with fixed‐angle rotor (Eppendorf 5415C or equivalent)
  • Tube rotator (capable of end‐over‐end inversion)
  • Pasteur pipet attached to a vacuum trap
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer.NOTE: All solutions should be ice cold and procedures should be carried out at 4°C or on ice.

Alternate Protocol 1: Immunoprecipitation Using Adherent Cells Lysed with a Nondenaturing Detergent Solution

  • Unlabeled or labeled cells grown as a monolayer on a tissue culture plate (unit 8.12)
  • Rubber policeman
NOTE: All solutions should be ice cold and procedures should be carried out at 4°C or on ice.

Alternate Protocol 2: Immunoprecipitation Using Cells Lysed with Detergent Under Denaturing Conditions

  • recipeDenaturing lysis buffer (see recipe)
  • Heating block set at 95°C (Eppendorf Thermomixer 5436 or equivalent)
  • 25‐G needle attached to 1‐ml syringe

Alternate Protocol 3: Immunoprecipitation Using Cells Lysed Without Detergent

  • recipeDetergent‐free lysis buffer (see recipe)
  • 25‐G needle attached to 3‐ml syringeNOTE: All solutions should be ice‐cold and procedures should be carried out at 4°C or on ice.

Alternate Protocol 4: Immunoprecipitation Using Yeast Cells Disrupted with Glass Beads

  • Unlabeled or radiolabeled yeast cells
  • Lysis buffer, ice cold: recipenondenaturing, recipedenaturing, or recipedetergent‐free lysis buffer (see reciperecipes)
  • Glass beads (acid‐washed, 425‐ to 600‐µm diameter; Sigma)
NOTE: All solutions should be ice‐cold and procedures should be carried out at 4°C or on ice.

Alternate Protocol 5: Immunoprecipitation with Antibody‐Sepharose

  Materials
  • Unlabeled cells, surface‐labeled cells (e.g., with 125I or biotin; unit 8.16) or biosynthetically 35S‐, 3H‐, or 14C‐labeled cells (unit 8.12)
  • recipeTriton X‐100 lysis buffer (see recipe)
  • recipeDilution buffer (see recipe)
  • Antibody (Ab)‐Sepharose (see protocol 7)
  • Activated, quenched (control) Sepharose, prepared as for Ab‐Sepharose (see protocol 7) but eliminating Ab or substituting irrelevant Ab during coupling
  • recipeTris/saline/azide (TSA) solution (see recipe)
  • 0.05 M Tris⋅Cl, pH 6.8 ( appendix 2A)
  • 2× SDS sample buffer (unit 8.4)
NOTE: Carry out all procedures in a 4°C cold room or on ice.

Support Protocol 1: Preparation of Antibody‐Sepharose

  Materials
  • 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; Amersham Pharmacia Biotech)
  • 0.2 M Na 2CO 3
  • recipeCyanogen bromide (CNBr)/acetonitrile (see recipe)
  • 1 mM and 0.1 mM HCl, ice‐cold
  • 0.05 M glycine (or ethanolamine), pH 8.0
  • recipeTris/saline/azide (TSA) solution (see recipe)
  • Dialysis tubing (molecular weight cutoff >10,000)
  • Whatman no. 1 filter paper
  • Buchner funnel
  • Erlenmeyer filtration flask
  • Water aspirator

Alternate Protocol 6: Immunoprecipitation of Radiolabeled Antigen with Anti‐Ig Serum

  • Normal serum
  • Anti‐Ig serum (Zymed Laboratories)
  • Antigen‐specific antiserum or antigen‐specific purified monoclonal antibody or antigen‐specific hybridoma culture supernatant

Basic Protocol 2: Immunoprecipitation‐Recapture

  Materials
  • recipeElution buffer (see recipe)
  • Beads containing bound antigen (see protocol 1, step )
  • 10% (w/v) BSA
  • recipeNondenaturing lysis buffer (see recipe)
  • Heating block set at 95°C (Eppendorf Thermomixer 5436 or equivalent)
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Figures

Videos

Literature Cited

Literature Cited
   Anderson, D.J. and Blobel, G. 1983. Immunoprecipitation of proteins from cell‐free translations. Methods Enzymol. 96:111‐120.
   Aruffo, A. 1998. Transient expression of proteins using COS cells. In Current Protocols in Molecular Biology (J.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.13.1‐16.13.7. John Wiley & Sons, New York.
   Cuatrecasas, P. 1970. Protein purification by affinity chromatography. J. Biol. Chem. 245:3059.
   Earl, P.L. and Moss, B. 1998. Characterization of recombinant vaccinia viruses and their products. In Current Protocols in Molecular Biology (J.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.18.1‐16.18.11. John Wiley & Sons, New York.
   Earl, P.L., Cooper, N., Wyatt, L.S., Moss, B., and Carroll, M.W. 1998a. Preparation of cell cultures and vaccinia stocks. In Current Protocols in Molecular Biology (J.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.16.1‐16.16.13. John Wiley & Sons, New York.
   Earl, P.L., Moss, B., Wyatt, L.S., and Carroll, M. 1998b. Generation of recombinant vaccinia virus. In Current Protocols in Molecular Biology (J.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.17.1‐16.17.19. John Wiley & Sons, New York.
   Franzusoff, A., Rothblatt, J., and Schekman, R. 1991. Analysis of polypeptide transit through yeast secretory pathway. Methods Enzymol. 194:662‐674.
   Gelb, W.G. 1973. Affinity chromatography: For separation of biological materials. Am. Lab. 81:61‐67.
   Gersten, D.M. and Marchalonis, J.J. 1978. A rapid, novel method for the solid‐phase derivatization of IgG antibodies for immune‐affinity chromatography. J. Immunol. Methods 24:305‐309.
   Harford, J. 1984. An artefact explains the apparent association of the transferrin receptor with a ras gene product. Nature 311:673‐675.
   Harlow, E. and Lane, D. 1999. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Irving, R.A., Hudson, P.J., and Goding, J.W. 1996. Construction, screening and expression of recombinant antibodies. In Monoclonal Antibodies: Principles and Practice (J.W. Goding, ed.). Academic Press, London.
   Kessler, S.W. 1975. Rapid isolation of antigens from cells with a staphylococcal protein A–antibody adsorbent: Parameters of the interaction of antibody‐antigen complexes with protein A. J. Immunol. 115:1617.
   Köhler, G. and Milstein, C. 1975. Continuous cultures of fused cells secreting antibody of predetermined specificity. Nature 256:495‐497.
   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.
   Moss, B. and Earl, P.L. 1998. Expression of proteins in mammalian cells using vaccinia viral vectors. In Current Protocols in Molecular Biology (J.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.15.1‐16.15.5. John Wiley & Sons, New York.
   Nisonoff, A. 1984. Introduction to Molecular Immunology. Sinauer Associates, Sunderland, Mass.
   Rapley, R. 1995. The biotechnology and applications of antibody engineering. Mol. Biotech. 3:139‐154.
Key References
   Harlow and Lane, 1988. See above.
  The above references describe various detergents and solubilization conditions.
   Helenius, A., McCaslin, D.R., Fries, E., and Tanford, C. 1979. Properties of detergents. Methods Enzymol. 56:734‐749.
   Hjelmeland, J.M. and Chrambach, A. 1984. Solubilization of functional membrane proteins. Methods Enzymol. 104:305‐318.
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