Generation and Utilization of Phosphorylation State–Specific Antibodies to Investigate Signaling Pathways

John A. Alberta1, Rosalind A. Segal2

1 Harvard Medical School and Dana‐Farber Cancer Institute, Boston, Massachusetts, 2 Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.14
DOI:  10.1002/0471142301.ns0314s04
Online Posting Date:  May, 2001
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Abstract

Phosphorylation state‐specific antibodies can be of great use, for example, in studying individual steps within a given signal transduction pathway. This unit presents a general approach to the generation and purification of phosphorylation state‐specific antibodies. In addition to their ability to detect phosphorylation at a particular key site, these antibodies are often more sensitive for biochemical studies. Besides their application in immunoblotting procedures, activation state‐specific antibodies can be used as immunohistochemical reagents. Thus, critical changes in phosphorylation can be monitored as described on an individual cell basis or in fixed tissue sections. Such antibodies can be used to address fundamental questions about signal transduction pathways during physiologic events that cannot be resolved by more conventional methodologies.

     
 
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Table of Contents

  • Basic Protocol 1: Peptide Coupling and Rabbit Immunization to Produce Phosphorylation State–Specific Antibodies
  • Basic Protocol 2: Purification of Phosphorylation State–Specific Antibodies
  • Support Protocol 1: Preparation of Peptide and Phosphopeptide Affinity Columns
  • Basic Protocol 3: Analysis of Phosphorylation State–Specific Antibodies by Immunoblotting
  • Support Protocol 2: Preparation of Extracts for Antibody Analysis
  • Basic Protocol 4: Staining Cultured Cells with Phosphorylation State–Specific Antibodies
  • Basic Protocol 5: Staining Tissue Sections with Phosphorylation State–Specific Antibodies
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Peptide Coupling and Rabbit Immunization to Produce Phosphorylation State–Specific Antibodies

  Materials
  • Sep‐Pak syringe column (Millipore) and acetonitrile, 0.1% trifluoroacetic acid, and 70% acetonitrile (for desalting, if needed)
  • Peptide of interest containing N‐ or C‐terminal cysteine (custom synthesized)
  • Appropriate solvent (e.g., water or DMSO)
  • Maleimide‐activated carrier protein (e.g., maleimide‐activated KLH, Pierce)
  • 5‐ or 10‐ml desalting columns (e.g., Presto columns, Pierce; Biogel P6DG, Bio‐Rad)
  • Protein assay kit, such as dye‐binding assay (e.g., Bio‐Rad)
  • BSA (for use as protein standard)
  • 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB or Ellman's reagent; Sigma)
  • Rabbits (e.g., New Zealand White)
  • Complete and incomplete Freunds adjuvant (CFA and IFA; Sigma)
  • 3‐ml Luer‐Lok syringes
  • 25‐G (1‐in.) needles
  • Water bath, 37°C
  • 0.45‐µm syringe filter (e.g., Millipore)

Basic Protocol 2: Purification of Phosphorylation State–Specific Antibodies

  Materials
  • Protein A–Sepharose (e.g., Pharmacia Biotech)
  • 100 mM Tris⋅Cl, pH 8.0 ( appendix 2A)
  • Antiserum from immunized rabbit (see protocol 1)
  • 10 mM Tris⋅Cl, pH 8.0 ( appendix 2A), without and with 0.5 M NaCl (Tris⋅Cl/NaCl)
  • 100 mM glycine, pH 3.0
  • 2 M Tris⋅Cl, pH 8.0
  • Protein assay kit(s), such as Bradford assay (e.g., Pierce) and dye‐binding assay (e.g., Bio‐Rad)
  • Sodium azide (optional)
  • Immobilized carrier protein (KLH, OVA, or BSA) column (see protocol 3 for preparation; optional)
  • Unphosphorylated peptide column (see protocol 3)
  • Phosphopeptide column (see protocol 3)
  • Phosphotyrosine, phosphoserine, or phosphothreonine column (see protocol 3)
  • 100 mM triethylamine, pH 11.5 (prepared fresh weekly)
  • Gentle elution buffer (Pierce)
  • BSA (e.g., Sigma)
  • Empty 3 × 20–cm fritted chromatographic column (30‐ml bed volume; e.g., Pharmacia Biotech)
  • Peristaltic pump
  • Centriprep concentrators (Amicon; optional)
  • Additional reagents and equipment for immunoblotting (see protocol 4) and immunostaining (see protocol 6 and protocol 75)
NOTE: Perform all steps at 4°C.

Support Protocol 1: Preparation of Peptide and Phosphopeptide Affinity Columns

  Materials
  • Peptide (phosphopeptide, unphosphorylated peptide, or carrier protein) or phosphoamino acid (phosphotyrosine, phosphoserine, or phosphothreonine) for immobilization on column
  • DMSO (anhydrous)
  • 100% ethanol
  • Activated column material, such as Affi‐Gel‐10 for basic peptides, or Affi‐Gel‐15 for acidic peptides (Bio‐Rad)
  • Isopropanol
  • 100 mM ethanolamine
  • 100 mM Tris⋅Cl, pH 8.0 ( appendix 2A) with and without 0.02% sodium azide
  • Empty 5‐ or 10‐ml chromatographic column

Basic Protocol 3: Analysis of Phosphorylation State–Specific Antibodies by Immunoblotting

  Materials
  • Membrane‐ or filter‐bound samples, with positive and negative controls (see protocol 5)
  • TBST (see recipe), without and with 2% (w/v) BSA or 5% (w/v) nonfat dry milk
  • Primary antibody: purified rabbit phosphorylation state–specific antibody (see Basic Protocols protocol 11 and protocol 22)
  • Secondary antibody conjugated to the enzyme used to produce the chromophore for detection (e.g., horseradish peroxidase or alkaline phosphatase; e.g., Promega, Bio‐Rad)
  • Blot development kits (e.g., ABC kit, Vector Labs, or others from Schleicher and Shuell, ECL, Amersham, or Promega)
  • Film (e.g., Kodak XARS X‐ray film, for enhanced chemiluminescence detection)

Support Protocol 2: Preparation of Extracts for Antibody Analysis

  Materials
  • Cells or tissue samples
  • TBS (see recipe), 4°C
  • NP‐40 extraction buffer (see recipe), 4°C
  • Protein assay kit, such as dye‐binding assay (Bio‐Rad)
  • 1‐ to 2‐ml Dounce homogenizer with loose‐fitting type B pestle (for tissue samples)

Basic Protocol 4: Staining Cultured Cells with Phosphorylation State–Specific Antibodies

  Materials
  • Cells of interest grown on coverslips
  • TBSV (see recipe), with and without 4% (v/v) paraformaldehyde (unit 1.1)
  • Primary antibody: purified rabbit phosphorylation state–specific antibody (see protocol 1 and protocol 22)
  • TBS (see recipe) with 2% normal serum (species‐matched to primary antibody)
  • Secondary antibody–fluorophore (e.g., horseradish peroxidase or alkaline phosphatase) conjugate
  • TBST (see recipe)
  • Mounting medium
  • Microscope slides
  • Fluorescent microscope

Basic Protocol 5: Staining Tissue Sections with Phosphorylation State–Specific Antibodies

  Materials
  • Animal to be used as tissue source
  • TBS, PBS, or TBSV (see reciperecipes) with 4% (v/v) paraformaldehyde (unit 1.1)
  • 30% (w/v) sucrose
  • Embedding medium (e.g.,Tissue Tek)
  • recipeTBS with 5% normal serum (species‐matched to primary antibody) and 0.5% NP‐40
  • Primary antibody: purified rabbit phosphorylation state–specific antibody (see Basic Protocols protocol 11 and protocol 22)
  • TBSV (see recipe), with and without 2% normal goat serum
  • Secondary antibody
  • 0.03% (v/v) hydrogen peroxide (DAB/H 2O 2) with and without 1 mg/ml diaminobenzidine (DAB)
  • Secondary antibody–fluorophore conjugate (e.g., Cy3–goat anti‐rabbit; for immunofluorescence)
  • Perfusion equipment (peristaltic pump, 18‐G needle, 1‐mm‐i.d. flexible tubing, and scissors suitable for opening the animal), sterile
  • ABC kit (Vector Labs)
  • Cryostat (see unit 1.1)
  • Additional reagents and equipment for tissue preparation and fixation (unit 1.1)
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Figures

  •   FigureFigure 3.14.1 The activation state–specific pY490 antibody is more sensitive than conventional reagents for detecting Trk phosphorylation. Cell extracts were prepared from control (−) or BDNF‐treated (+) primary cerebellar neuron cultures from P4 mice and then processed as follows. Left‐hand immunoblot: lysate (500 µg) was immunoprecipitated (ip) using a pan‐Trk antibody that recognizes all three Trk family members. The immunoprecipitate was size fractionated on an SDS‐polyacrylamide gel and immunoblotted with a commercially available monoclonal antibody to phosphotyrosine (PY). Right‐hand immunoblot: lysate (50 µg) was size fractionated on an SDS‐polyacrylamide gel and immunoblotted with pY490 antibody.
  •   FigureFigure 3.14.2 Localization of activated neurotrophin receptor in the cerebellum. P14 mouse brains were fixed, sectioned, and processed for immunohistochemistry with the pY490 antibody as described in . (A, B) Cerebellar sections at low and high magnification (scale bars equal 100 mm and 25 mm, respectively). Bold arrow indicates a Purkinje cell body. Fine arrows indicate individual granule cells in the molecular and internal granule cell layers. (CE) Same as panels A and B except that the indicated peptides or phosphopeptides were premixed with the pY490 antibody to establish staining specificity. Scale bar for C through E equals 50 mm. (F) Mouse cerebellar extract (P8) was size fractionated on SDS‐polyacrylamide gels and immunoblotted with pY490. Arrow indicates phosphorylated Trk.

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Literature Cited

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