Production of Antibodies That Recognize Specific Tyrosine‐Phosphorylated Peptides

Michael P. DiGiovanna1, Robert R. Roussel2, David F. Stern1

1 Yale University School of Medicine, New Haven, Connecticut, 2 Dartmouth College, Hanover, New Hampshire
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 16.6
DOI:  10.1002/0471143030.cb1606s13
Online Posting Date:  February, 2002
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Abstract

Phosphorylation of tyrosine residues of various proteins plays an important role in regulation of the functional activities of proteins within the cell. Antibodies to specific tyrosine‐phosphorylated peptides are a valuable tool for analysis of the localization and function of individual phosphoproteins. This unit describes the methods used to produce and screen monoclonal and polyclonal antibodies for specificity for only the phosphorylated state of a peptide.

     
 
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Table of Contents

  • Basic Protocol 1: Production of Polyclonal Anti‐Phosphopeptide Antibodies
  • Basic Protocol 2: Production of Monoclonal Anti‐Phosphopeptide Antibodies
  • Support Protocol 1: Synthesis of Peptides
  • Support Protocol 2: Coupling of Peptides to Affi‐Gel 10 Affinity Matrix
  • Support Protocol 3: Coupling of Phosphotyrosine to Affi‐ Gel 10 Affinity Matrix
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Production of Polyclonal Anti‐Phosphopeptide Antibodies

  Materials
  • BSA‐agarose affinity matrix (Sigma) packed (see protocol 4)in a 10‐ml bed volume column
  • Phosphotyrosine affinity matrix column (10‐ml bed volume; see protocol 5)
  • Crude serum from rabbit immunized with phosphopeptide‐BSA conjugate (refer to Strategic Planning, above)
  • PBS/azide: PBS appendix 2A) containing 0.02% (w/v) sodium azide (store indefinitely at 4°C or room temperature)
  • Cognate nonphosphopeptide affinity matrix column (3‐ml bed volume; see protocol 3 and protocol 4 and Strategic Planning
  • 3 M NaSCN
  • Homologous phosphopeptide affinity matrix columns (optional; 3‐ml bed volume; see protocol 3 and protocol 4 and Strategic Planning)
  • Positive‐selection phosphopeptide affinity matrix column (3‐ml bed volume; see protocol 3 and protocol 4 and Strategic Planning)
  • 3.5 M and 4.5 M MgCl 2 (optional; see Critical Parameters)
  • Spectrophotometer (optional)
  • Dialysis tubing (MWCO 12,000 to 14,000; 10 mm width, 6.4 mm diameter; e.g., Spectra/Por 4 from Spectrum)
  • Additional reagents and equipment for dialysis ( appendix 3C), protein quantitation ( appendix 3B), and analysis of antibodies by ELISA ( appendix 3A) or immunoblotting (unit 6.2)

Basic Protocol 2: Production of Monoclonal Anti‐Phosphopeptide Antibodies

  Materials
  • Candidate hybridoma cell lines from fusion (refer to Strategic Planning and unit 16.1)
  • HT medium; prepare as for HAT medium (unit 1.2) except omit aminopterin
  • Screening diluent (see recipe)
  • BSA‐conjugated cognate phosphopeptide (for use as ELISA antigen; refer to Strategic Planning and protocol 3)
  • Preimmune serum from mouse used to produce hybridoma line (negative control)
  • Immune serum from mouse used to produce hybridoma line (positive control)
  • BSA‐conjugated cognate nonphosphopeptide (for use as ELISA antigen; refer to Strategic Planning and protocol 3)
  • BSA‐conjugated noncognate phosphotyrosyl peptide (for use as ELISA antigen; refer to Strategic Planning and protocol 3)
  • BSA‐conjugated homologous phosphopeptides (optional, for use as ELISA antigens; refer to Strategic Planning and protocol 3)
  • 96‐well polystyrene tissue culture plates
  • 96‐grid note sheets
  • Additional reagents and equipment for cloning by limiting dilution, ELISA, and freezing and recovery of hybridoma cell lines ( appendix 3A)
NOTE: All solutions and equipment coming into contact with live cells must be sterile, and proper asceptic technique should be used accordingly.

Support Protocol 1: Synthesis of Peptides

  Materials
  • Synthetic oligopeptide for coupling (see protocol 3)
  • Dimethyl sulfoxide (DMSO)
  • N‐methylmorpholine (99% purity; Acros Organics)
  • Affi‐Gel 10 (Bio‐Rad) or equivalent activated support matrix
  • Ethanolamine
  • 0.1 M ethanolamine⋅HCl, pH 8.0
  • High‐salt/high‐pH solution: 0.5 M NaCl/0.4% (w/v) sodium bicarbonate
  • High‐salt/low‐pH solution: 0.5 M NaCl/100 mM sodium acetate, pH 4.2
  • PBS/azide:PBS ( appendix 2A) containing 0.02% (w/v) sodium azide
  • 0.5 M NaCl
  • 3 M NaSCN
  • Polypropylene screw‐cap centrifuge tubes (do not use polystyrene tubes with DMSO)
  • End‐over‐end rotator
  • IEC Clinical centrifuge (or equivalent)
  • Vacuum aspirator
  • Glass chromatography columns, ≥5‐ml capacity

Support Protocol 2: Coupling of Peptides to Affi‐Gel 10 Affinity Matrix

  • Phosphotyrosine
  • 0.4% (w/v) sodium bicarbonate
  • 1 M NaOH (optional)
  • Fritted‐glass funnel and vacuum aspirator
  • Glass chromatography column, ≥14‐ml capacity
  • Additional reagents and equipment for coupling peptides to Affi‐Gel 10 affinity matrix (see protocol 4)
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Figures

Videos

Literature Cited

Literature Cited
   Bangalore, L., Tanner, A.J., Laudano, A.P., and Stern, D.F. 1992. Antiserum raised against a synthetic phosphotyrosine‐containing peptide selectively recognizes p185neu/erbB‐2 and the epidermal growth factor receptor. Proc. Natl. Acad. Sci. U.S.A. 89:11637‐11641.
   Barany, G. and Merrifield, B. 1979. Solid phase peptide synthesis. In The Peptides, Vol. I (E. Gross and J. Meienhofer eds.) pp. 1‐284. Academic Press, New York.
   Chang, C.D. and Meienhofer, J. 1978. Solid‐phase peptide synthesis using mild base cleavage of N alpha‐fluorenylmethoxycarbonylamino acids, exemplified by a synthesis of dihydrosomatostatin. Int. J. Pept. Protein Res. 11: 246‐249.
   Czernik, A.J., Girault, J.‐A., Nairn, A.C., Chen, J., Snyder, G., Kebabian, J., and Greengard, P. 1991. Production of phosphorylation state–specific antibodies. Methods Enzymol. 201: 264‐283.
   DiGiovanna, M.P. and Stern, D.F. 1995. Activation state–specific monoclonal antibody detects tyrosine phosphorylated p185neu/erbB‐2 in a subset of human breast tumors overexpressing this receptor. Cancer Res. 55:1946‐1955.
   Doolittle, R.F. 1986. Of urfs and orfs: A primer on how to analyze derived amino acid sequences. University Science Books, Mill Valley, Calif.
   Epstein, R.J. 1995. Preferential detection of catalytically inactive c‐erbB‐2 by antibodies to unphosphorylated peptides mimicking receptor tyrosine autophosphorylation sites. Oncogene 11: 315‐323.
   Epstein, R.J., Druker, B.J., Roberts, T.M., and Stiles, C.D. 1992. Synthetic phosphopeptide immunogens yield activation‐specific antibodies to the c‐erbB‐2 receptor. Proc. Natl. Acad. Sci. U.S.A. 89:10435‐10439.
   Frackelton, A.R.Jr., Ross, A.H., and Eisen, H.N. 1983. Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus‐transformed cells and growth factor–stimulated cells. Mol. Cell. Biol. 3: 1343‐1352.
   Kawakatsu, H., Sakai, T., Takagaki, Y., Shinoda, Y., Saito, M., Owada, M.K., and Yano, J. 1996. A new monoclonal antibody which selectively recognizes the active form of src tyrosine kinase. J. Biol. Chem. 271:5680‐5685.
   Kitas, E., Kung, E., and Bannwarth, W. 1994. Chemical synthesis of O‐thiophosphotyrosyl peptides. Int. J. Pept. Protein Res. 43:146‐153.
   Nairn, A.C., Detre, J.A., Casnellie, J.E., and Greengard, P. 1982. Serum antibodies that distinguish between the phospho‐ and dephospho‐forms of a phosphoprotein. Nature 299:734‐736.
   Ross, A.H., Baltimore, D., and Eisen, H.N. 1981. Phosphotyrosine‐containing proteins isolated by affinity chromatography with antibodies to a synthetic hapten. Nature 294:654‐656.
   Roussel, R. 1990. Synthetic phosphopeptides modeled on the carboxyl terminus of pp60C‐SRC: Specific antibodies, binding and effects on kinase activity. M.S. thesis, University of New Hampshire, Durham.
   Stern, D.F. 1991. Antiphosphotyrosine antibodies in oncogene and receptor research. Methods Enzymol. 198:494‐501.
   Tzartos, S.J., Kouvatsou, R., and Tzartos, E. 1995. Monoclonal antibodies as site‐specific probes for the acetylcholine‐receptor δ‐subunit tyrosine and serine phosphorylation sites. Eur. J. Biochem. 228:463‐472.
Key References
   Bangalore et al., 1992. See above
  A description of the production of polyclonal anti‐phosphopeptide antibody by the method described in this unit.
   Czernik et al., 1991. See above.
  A general discussion of the production of phosphorylation‐dependent antibodies.
   DiGiovanna and Stern, 1995. See above.
  A description of the production of monoclonal anti‐phosphopeptide antibody by the method described in this unit.
   Doolittle, 1986. See above.
  A general discussion of the analysis of protein sequences and the production of anti‐peptide antibodies.
   Epstein et al., 1992. See above.
  A description of the production of polyclonal anti‐phosphopeptide antibody by a variation of the method described in this unit.
   Harlow, E. and Lane, D. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  A comprehensive text describing the production and utilization of antibodies.
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