Immunological Detection of Phosphorylation

Zhong Yao1, Rony Seger1

1 The Weizmann Institute of Science, Rehovot
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.2
DOI:  10.1002/0471143030.cb1402s00
Online Posting Date:  May, 2001
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Abstract

Incorporation of phosphates into serine, threonine, and tyrosine acceptors in proteins is a common mechanism for regulating protein function. This unit presents protocols that use specific anti‐phosphoamino acid (PAA) and anti‐phosphoprotein antibodies to detect protein phosphorylation and protein kinase activity. Immunoblotting to detect protein phosphorylation using either anti‐PAA or anti‐phosphoprotein antibodies. This is a convenient method that usually yields impressive results. Phosphorylation can also be detected by immunoprecipitation followed by immunoblot analysis or by immunofluorescent staining; these methods are typically more complicated and time consuming. All three methods have been successfully used to detect protein phosphorylation with a wide variety of antibodies and most phosphorylated proteins.

     
 
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Table of Contents

  • Basic Protocol 1: Immunodetection of Protein Phosphorylation by Immunoblotting
  • Basic Protocol 2: Immunodetection of Protein Phosphorylation by Immunoprecipitation Followed by Immunoblotting
  • Basic Protocol 3: Fluorescent Immunostaining of Tissue Culture Cells
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Immunodetection of Protein Phosphorylation by Immunoblotting

  Materials
  • Rat1 cells
  • Dulbecco's modified Eagle medium containing 10% heat‐inactivated fetal bovine serum (DMEM/10% FBS; appendix 2A)
  • Starvation medium: DMEM/0.1% FBS
  • Epidermal growth factor (EGF) buffer: phosphate‐buffered saline (PBS)/0.5 mg/ml bovine serum albumin (BSA, crystalline)
  • 50 µg/ml EGF in EGF buffer
  • PBS ( appendix 2A), ice cold
  • Homogenization buffer (see recipe), ice cold
  • Lysis buffer: recipehomogenization buffer/1% Triton X‐100, ice cold
  • Kinase buffer (see recipe), ice cold
  • Coomassie protein assay reagent (Pierce)
  • Protein standards: 5, 10, 20, 50, 100, and 200 µg/ml BSA in recipehomogenization buffer/0.03% Triton X‐100
  • 4× SDS‐polyacrylamide gel electrophoresis (PAGE) sample buffer (see recipe)
  • Prestained protein markers (16 to 200 kDa)
  • 12% SDS‐polyacrylamide gel (12% polyacrylamide/0.32% bisacrylamide; unit 6.1)
  • Transfer buffer: 50 mM Tris⋅Cl (pH ∼8.8)/50 mM glycine
  • Tris‐buffered saline/Tween 20 (TBST; see recipe)
  • Blocking solution: recipeTBST/2% BSA
  • Primary antibodies: monoclonal anti–active MAP kinase and polyclonal anti–general MAP kinase
  • Secondary antibodies: horseradish peroxidase (HRP)–conjugated goat anti–rabbit and alkaline phosphatase (AP)–conjugated goat anti‐mouse
  • AP detection system (e.g., Promega)
  • Enhanced chemiluminescence (ECL) detection system (see recipe)
  • 6‐cm tissue culture plates
  • 1.5‐ml microcentrifuge tubes, prechilled to 4°C (four sets of six, each set labeled 1 to 6)
  • Stopwatch
  • 1‐ml pipet tips, prechilled to 4°C
  • Microcentrifuge, 4°C
  • 96‐well flat‐bottomed microtiter plate
  • Microtiter plate reader, 595‐nm wavelength
  • Nitrocellulose membrane cut to size of gel
  • Whatman 3MM filter paper, two pieces, cut to size of gel
  • Transfer apparatus for electroblots (e.g., Bio‐Rad)
  • Flat container for washing nitrocellulose membrane
  • Additional reagents and equipment for tissue culture (unit 1.1), chromogenic and luminescent visualization (unit 6.2), and SDS‐PAGE (unit 6.1)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All tissue culture incubations should be performed in a humidified 37°C, 5% to 10% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Immunodetection of Protein Phosphorylation by Immunoprecipitation Followed by Immunoblotting

  Materials
  • Protein A–Sepharose beads
  • PBS ( appendix 2A), room temperature and ice cold
  • Antibodies for immunoprecipitation of the desired protein (1 to 5 µg per reaction)
  • Homogenization buffer (see recipe), ice cold
  • Cellular extract in lysis buffer (see protocol 1, step )
  • RIPA buffer (see recipe), ice cold
  • 0.5 M LiCl solution, ice cold
  • 0.1 M Tris⋅Cl, pH 8.0 ( appendix 2A)
  • 4× SDS‐PAGE sample buffer (see recipe)
  • 12% SDS‐polyacrylamide gel (12% polyacrylamide/0.32% bisacrylamide; unit 6.1)
  • End‐over‐end rotator
  • Microcentrifuge, 4°C
  • Additional reagents and equipment for analysis by SDS‐PAGE and immunoblotting (see protocol 1 and unit 6.1)

Basic Protocol 3: Fluorescent Immunostaining of Tissue Culture Cells

  Materials
  • Rat1 cells
  • DMEM/10% FBS ( appendix 2A)
  • Starvation medium: DMEM/0.1% FBS
  • PBS ( appendix 2A)
  • 3% (w/v) paraformaldehyde in PBS
  • Permeabilization buffer: PBS/0.2% (v/v) Triton X‐100
  • Primary antibody
  • Secondary antibodies: fluorescein isothiocyanate (FITC)–conjugated goat anti‐rabbit and rhodamine‐conjugated goat anti–mouse
  • 6‐well tissue culture plates
  • 22‐mm‐square coverslips, sterile
  • Parafilm, two 10 × 20–cm pieces
  • Slide mounting medium (e.g., Polymount, Polyscience)
  • Confocal or fluorescent microscope
  • Additional reagents and equipment for tissue culture (unit 1.1)
NOTE: All solutions and equipment coming into contact with cells must be aseptic, and proper aseptic technique should be used accordingly.NOTE: All tissue culture incubations should be performed in a humidified 37°C, 5% to 10% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.
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Figures

Videos

Literature Cited

Literature Cited
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   Campbell, J.S., Seger, R., Graves, J.D., Graves, L.M., Jensen, A.M., and Krebs, E.G. 1995. The MAP kinase cascade. Recent Prog. Horm. Res. 50:131‐159.
   Comoglio, P.M., Di Renzo, M.F., Tarone, G., Giancotti, F.G., Naldini, L., and Marchisio, P.C. 1984. Detection of phosphotyrosine‐containing proteins in the detergent‐insoluble fraction of RSV–transformed fibroblasts by azobenzene phosphonate antibodies. EMBO J. 3:483‐489.
   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.
   Gabay, L., Seger, R., and Shilo, B.Z. 1997. In situ activation pattern of Drosophila EGF receptor pathway during development. Science 277:1103‐1106.
   Ginty, D.D., Kornhauser, J.M., Thompson, M.A., Bading, H., Mayo, K.E., Takahashi, J.S., and Greenberg, M.E. 1993. Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock. Science 260:238‐241.
   Heffetz, D., Fridkin, M., and Zick, Y. 1989. Antibodies directed against phosphothreonine residues as potent tools for studying protein phosphorylation. Eur. J. Biochem. 182:343‐348.
   Hunter, T. 1994. 1001 protein kinases redux—Towards 2000. Semin. Cell Biol. 5:367‐376.
   Hunter, T. 1995. Protein kinases and phosphatases: The yin and yang of protein phosphorylation and signaling. Cell 80:225‐236.
   Karin, M. and Hunter, T. 1995. Transcriptional control by protein phosphorylation: Signal transmission from the cell surface to the nucleus. Curr. Biol. 5:747‐757.
   Krebs, E.G. 1994. The growth of research on protein phosphorylation. Trends Biochem. Sci. 19:439.
   Marshall, C.J. 1996. Ras effectors. Curr. Opin. Cell Biol. 8:197‐204.
   Pang, D.T., Sharma, B.R., Shafer, J.A., White, M.F., and Kahn, C.R. 1985. Predominance of tyrosine phosphorylation of insulin receptors during the initial response of intact cells to insulin. J. Biol. Chem. 260:7131‐7136.
   Payne, D.M., Rossomando, A.J., Martino, P., Erickson, A.K., Her, J.‐H., Shabanowitz, J., Hunt, D.F., Weber, M.J., and Sturgill, T.W. 1991. Identification of the regulatory phosphorylation sites in pp42/mitogen‐activated protein kinase (MAP kinase). EMBO J. 10:885‐892.
   Robbins, D.J. and Cobb, M.H. 1992. Extracellular signal–regulated kinases 2 autophosphorylates on a subset of peptides phosphorylated in intact cells in response to insulin and nerve growth factor: Analysis by peptide mapping. Mol. Biol. Cell 3:299‐308.
   Robinson, M.J. and Cobb, M.H. 1997. Mitogen‐activated protein kinase pathways. Curr. Opin. Cell Biol. 9:180‐186.
   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.
   Salinovich, O. and Montelaro, R.C. 1986. Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Anal. Biochem. 156:341‐347.
   Seger, R. and Krebs, E.G. 1995. The MAP kinase signaling cascade. FASEB J. 9:726‐735.
   Seger, R., Seger, D., Reszka, A.A., Munar, E.S., Eldar‐Finkelman, H., Dobrowolska, G., Jensen, A.M., Campbell, J.S., Fischer, E.H., and Krebs, E.G. 1994. Over‐expression of mitogen‐activated protein kinase kinase (MAPKK) and its mutants in NIH‐3T3 cells: Evidence that MAPKK's involvement in cellular proliferation is regulated by phosphorylation of serine residues in its kinase subdomains VII and VIII. J. Biol. Chem. 269:29876‐29886.
   Volberg, T., Zick, Y., Dror, R., Sabanay, I., Gilon, C., Levitzki, A., and Geiger, B. 1992. The effect of tyrosine‐specific protein phosphorylation on the assembly of adherens‐type junctions. EMBO J. 11:1733‐1742.
   Yung, Y., Dolginov, Y., Yao, Z., Rubinfeld, H., Michael, D., Hanoch, T., Roubini, E., Lando, Z., Zharhary, D., and Seger, R. 1997. Detection of ERK activation by a novel monoclonal antibody. FEBS Lett. 408:292‐296.
Key Reference
   Gabay et al., 1997. See above.
  Reports the use of anti–active MAP kinase antibodies (anti‐diphospho‐ERK) to follow MAP kinase (ERK) activation during different stages of Drosophila development, with immunostaining and immunoblotting both used throughout the study.
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