Preparation and Application of Polyclonal and Monoclonal Sequence‐Specific Anti‐Phosphoamino Acid Antibodies

Tong Sun1, Ralph B. Arlinghaus1

1 University of Texas M.D. Anderson Cancer Center, Houston, Texas
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 13.6
DOI:  10.1002/0471140864.ps1306s34
Online Posting Date:  February, 2004
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Abstract

This unit discusses the issues that must be considered in the design, production, and characterization of polyclonal and monoclonal sequence‐specific anti‐phosphoamino acid antibodies. Protocols are provided for generating and purifying such antibodies, and methods are also provided for producing useful polyclonal antibodies in a non‐purified form. Support protocols describe coupling of peptides or phosphotyrosine to a solid support for use in affinity chromatography. An example of the generation, purification, and characterization of two sequence‐specific anti‐phosphopeptide antibodies specific for different sequences of a single phosphoprotein is described. The cross‐reactivity of such antibodies, which is a common problem with anti‐peptide antibodies, is also discussed.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Affinity Purification of Polyclonal Sequence‐Specific Anti‐Phosphopeptide Antibodies
  • Basic Protocol 2: Production of Mouse Monoclonal Sequence‐Specific Anti‐Phosphopeptide Antibodies
  • Support Protocol 1: Coupling Peptides to Affi‐Gel 10 Matrix
  • Support Protocol 2: Coupling Phosphotyrosine to an Affi‐Gel Matrix
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Affinity Purification of Polyclonal Sequence‐Specific Anti‐Phosphopeptide Antibodies

  Materials
  • BSA‐agarose affinity matrix (Sigma) packed (see protocol 3) in a 10‐ml bed volume column
  • Phosphotyrosine affinity matrix column (10‐ml bed volume; see protocol 4), if an anti‐phosphotyrosine antibody is being made
  • Clarified, crude antiserum from rabbit immunized with phosphopeptide‐carrier conjugate
  • PBS/azide: PBS ( appendix 2E) containing 0.02% (w/v) sodium azide (store indefinitely at 4°C or room temperature)
  • 3 M NaSCN
  • Cognate nonphosphopeptide affinity matrix column (3‐ml bed volume; see protocol 3 and Strategic Planning)
  • Homologous phosphopeptide affinity matrix columns (optional; 3‐ml bed volume; see protocol 3 and Strategic Planning)
  • Positive‐selection phosphopeptide affinity matrix column (3‐ml bed volume; see protocol 3 and Strategic Planning)
  • 3.5 M and 4.5 M MgCl 2 (optional)
  • 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 (unit 4.4), protein quantitation (units 3.1& 3.4), and analysis of antibodies by ELISA or immunoblotting (unit 10.10)

Basic Protocol 2: Production of Mouse Monoclonal Sequence‐Specific Anti‐Phosphopeptide Antibodies

  Materials
  • Candidate hybridoma cell lines from fusion
  • HT medium (see recipe)
  • Screening diluent (see recipe)
  • BSA‐conjugated cognate phosphopeptide (for use as ELISA antigen; see protocol 3 and unit 18.3)
  • Pre‐immune 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; unit 18.3)
  • BSA‐conjugated non‐cognate phosphotyrosyl peptide (for use as ELISA antigen; unit 18.3)
  • BSA‐conjugated homologous phosphopeptides (optional, for use as ELISA antigens; unit 18.3)
  • 96‐well polystyrene tissue culture plates
  • Grid note sheets
  • Additional reagents and equipment for cloning by limiting dilution, ELISA, and freezing and recovery of hybridoma cell lines
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

Support Protocol 1: Coupling Peptides to Affi‐Gel 10 Matrix

  Materials
  • Synthetic oligopeptide for coupling (see Strategic Planning)
  • Dimethyl sulfoxide (DMSO)
  • N‐methylmorpholine (99% purity; Acros Organics)
  • Affi‐Gel 10 (Bio‐Rad) or equivalent activated support matrix
  • Ethanolamine
  • 0.1 M ethanolamine·Cl, 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 2E) 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 (Bio‐Rad)

Support Protocol 2: Coupling Phosphotyrosine to an Affi‐Gel Matrix

  Materials
  • Phosphotyrosine
  • 0.4% (w/v) sodium bicarbonate
  • 1 M NaOH (optional)
  • Fritted‐glass funnel and vacuum aspirator
  • Glass chromatography column, ≥14‐ml capacity (Bio‐Rad)
  • Additional reagents and equipment for coupling peptides to Affi‐Gel 10 affinity matrix (see protocol 3)
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Figures

Videos

Literature Cited

   Alberta, J.A. and Stiles, C.D. 1997. Phosphorylation‐directed antibodies in high flex screens for compounds that modulate signal transduction. BioTechniques 23:490‐493.
   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.
   Banin, S., Moyal, L., Shieh, S.‐Y., Taya, C., Anderson, C.W., Chessa, L., Smorodinsky, N.I., Prives, C., Reiss, Y., Shiloh, Y., and Ziv, Y. 1998. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281:1674‐1677.
   Bodnar, R.J., Gu, M., Li, Z., Englund, G.D., and Du, X. 1999. The cytoplasmic domain of the platelet glycoprotein Ib is phosphorylated at serine 609. J. Biol. Chem. 274:33474‐33479.
   Campbell, M.L. and Arlinghaus, R.B. 1991. Current status of the BCR gene and its involvement with human leukemia. Adv. Cancer Res. 57:227‐256.
   Canman, C.E., Lim, D.‐S., Cimprich, K.A., Taya, Y., Tamai, K., Sakaguchi, K., Appella, E.A., Kastan, M.B., and Siliciano, J.D. 1998. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281:1677‐1679.
   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. Dept. Protein Res. 11:246‐249.
   Coghlan, M.P., Pillay, T.S., Tavare, J.M., and Siddle, K. 1994. Site‐specific anti‐phosphopeptide antibodies: Use in assessing insulin receptor serine/threonine phosphorylation state and identification of serine‐1327 as a novel site of phorbol ester‐induced phosphorylation. Biochem. J. 303:893‐899.
   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.
   Daley, G.Q., van Etten, R.A., and Baltimore, D. 1990. Inductin of chronic myelogenous leukemia in mice by the P210bcr‐abl gene of the Philadelphia chromosome. Science 247:824‐830.
   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.
   Cooper, H.M. and Paterson, Y. 1995. Production of polyclonal antisera. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and, W. Strober, eds.) pp. 2.4.1‐2.4.9. John Wiley & Sons, New York.
   Guo, J.Q., Wang, J.Y.J., and Arlinghaus, R.B. 1991. Detection of BCR‐ABL proteins in blood cells of benign phase chronic myelogenous leukemia patients. Cancer Res. 51:3048‐3051.
   Hawk, N., Sun, T., Xie, S., Wang, Y., Wu, Y., Liu, J., and Arlinghaus, R. 2002. Inhibition of the Bcr‐Abl oncoprotein by Bcr requires phosphoserine 354. Cancer Res. 62:386‐390.
   Heisterkamp, N., Stam, K., Groffen, J., de Klein, A., and Grosveld, G. 1985. Structural organization of the bcr gene and its role in the Ph′translocation. Nature. 315:758‐761.
   Heisterkamp, N., Jenster, G., ten Hoeve, J., Zovich, D., Pattengale, P.K., and Groffen, J. 1990. Acute leukemia in Bcr‐Abl transgenic mice. Nature. 344:251‐253.
   Hornbeck, P., Fleisher, T.A., and Papadopoulos, N.M. 1991. Isotype determination of antibodies. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and, W. Strober, eds.) pp. 2.2.1‐2.2.6. John Wiley & Sons, New York.
   Ishida, R., Iwai, M., Marsh, K.L., Austin, C.A., Yano, T., Shibata, M., Nozaki, M., and Hara, A. 1996. Threonine‐1342 in human topoisomerase II alpha is phosphorylated throughout the cell cycle. J. Biol. Chem. 271: 30077‐30082.
   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.
   Kloetzer, W., Kurzrock, R., Smith, L., Talpaz, M., Spiller, M., Gutterman, J., and Arlinghaus, R. 1985. The human cellular abl gene product in the chronic myelogenous leukemia cell line K562 has an associated tyrosine protein kinase activity. Virology 140:230‐238.
   Konopka, J.B., Watanabe, S.M., and Witte, O.N. 1984. An alteration of the human c‐abl protein in K562 leukemia cells unmasks associated tyrosine kinase activity. Cell 37:1035‐1042.
   Liu, J., Campbell, M., Guo, J.Q., Xian, Y.M., Anderssen, B.S., and Arlinghaus, R.B. 1993. Bcr‐Abl tyrosine kinase is autophosphorylated or transphosphorylates P160 Bcr on tyrosine predominantly within the first BCR exon. Oncogene 8:101‐109.
   Liu, J., Wu, Y., Ma, G.Z., Lu, D., Haataja, L., Heisterkamp, N., Groffen, J., and Arlinghaus, R.B. 1996. Inhibition of Bcr serine kinase by tyrosine phosphorylation. Mol. Cell. Biol. 16:998‐1005.
   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.
   Ponticelli, A.S., Whitlock, C.A., Rosenberg, N., and Witte, O.N. 1982. In vivo tyrosine phosphorylations of the Abelson virus transforming protein are absent in its normal cellular homolog. Cell 29:953‐960.
   Puil, L., Liu, J., Gish, G., Mbamalu, G., Bowtell, D., Pelicci, P.G., Arlinghaus, R., and Pawson, T. 1994. Bcr‐Abl oncoproteins bind directly to activators of the Ras signalling pathway. EMBO J. 13:763‐773.
   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.
   Shtivelman, E., Lifshitz, B., Gale, R.P., and Canaani, E. 1985. Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature 315:550‐554.
   Smith, R.G., Dev, V.G., and Shannon, W.A. Jr. 1981. Characterization of a novel human pre‐B leukemia cell line. J. Immunol. 126:596‐602.
   Stam, K., Heisterkamp, N., Reynolds, F.H. Jr, and Groffen, J. 1987. Evidence that the phl gene encodes a 160,000‐dalton phosphoprotein with associated kinase activity. Mol. Cell. Biol. 7:1955‐1960.
   Stern, D.F. 1991. Antiphosphotyrosine antibodies in oncogene and receptor research. Methods Enzymol. 198:494‐501.
   Sun, T., Campbell, M., Gordon, W., and Arlinghaus, R.B. 2001. Preparation and application of antibodies to phosphoamino acid sequences. Biopolymers 60:61‐75.
   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.
   Wu, Y., Liu, J., and Arlinghaus, R.B. 1998. Requirement of two specific tyrosine residues for the catalytic activity of Bcr serine/theronine kinase. Oncogene 16:141‐146.
   Wu, Y., Ma, G., Lu, D., Lin, F., Xu, H‐J., Liu, J., and Arlinghaus, R.B. 1999. Bcr: A negative regulator of the Bcr‐Abl oncoprotein. Oncogene 18:4416‐4424.
   Zhang, H., Zha, X., Tan, Y., Hornbeck, P.V., Mastrangelo, A.J., Alessi, D.R., Polakiewicz, R.D., and Comb, M.J. 2002. Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs. J. Biol. Chem. 277:39379‐39387.
Key References
   Sun et al., 2001 (see above)
  A description of the production of polyclonal anti‐phosphopeptide antibody by the method described in this unit.
   DiGiovanna, M.P., Stern, D.F., and Roussel, R.R. 2003. Production of antibodies that recognize specific tyrosine‐phosphorylated peptides. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and, W. Strober, eds.) pp. 11.6.1‐11.6.19. John Wiley & Sons, New York.
  Source of and and .
   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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