Biochemical Fractionation of Brain Tissue for Studies of Receptor Distribution and Trafficking

Penelope J. Hallett1, Tiffany L. Collins1, David G. Standaert1, Anthone W. Dunah1

1 MassGeneral Institute for Neurodegenerative Disease (MIND), Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.16
DOI:  10.1002/0471142301.ns0116s42
Online Posting Date:  January, 2008
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Abstract

An important tool for studying the regulation of synapses is a rapid and reliable means of separating synaptic and intracellular proteins. This unit presents a technique for analysis of brain tissue which relies on differential centrifugation to separate proteins present at synaptic sites from those found in intracellular cytoplasmic and vesicular pools. The method is efficient in that only small amounts of tissue, such as might be obtained from a small region of a rodent brain, are required. It is reproducible and, in conjunction with immunoblot or immunoprecipitation techniques, can produce reliable quantitative data. The protocol will be of interest to those conducting a variety of different studies related to the localization and trafficking of brain receptors and signaling molecules. Curr. Protoc. Neurosci. 42:1.16.1‐1.16.16. © 2008 by John Wiley & Sons, Inc.

Keywords: subcellular; trafficking; immunoprecipitation; receptor; phosphorylation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Subcellular Fractionation of Brain Tissues
  • Support Protocol 1: Non‐Denaturing Protein Solubilization for Coimmunoprecipitation of Protein Complexes
  • Support Protocol 2: Denaturing Protein Solubilization for Immunoprecipitation Studies of Protein Phosphorylation
  • Support Protocol 3: DSP‐Mediated Cross‐Linking of Proteins for Coimmunoprecipitation Studies
  • Support Protocol 4: Immunoprecipitation of Phosphorylated Proteins and Coimmunoprecipitation of Protein Complexes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Subcellular Fractionation of Brain Tissues

  Materials
  • Laboratory rats (either gender)
  • Chilled isopentane (−35°C to −45°C)
  • TEVP buffer, 4°C (see recipe)
  • TEVP buffer containing 320 mM sucrose, 4°C (see recipe)
  • TEVP buffer containing 35.6 mM sucrose, 4°C (see recipe)
  • 4× sample buffer (see recipe)
  • SDS‐acrylamide gels (CPMB UNIT )
  • Broad‐range molecular weight markers
  • Polyvinylidene difluoride (PVDF) membrane (e.g., BioRad PVDF immunoblotting membrane)
  • TBST buffer (see recipe)
  • Non‐fat milk powder
  • Primary antibodies against antigens of interest
  • 0.1% sodium azide
  • HRP‐conjugated secondary antibodies
  • Chemiluminescent detection reagent (e.g., Western Lightning from Perkin‐Elmer)
  • Tissue homogenizer (e.g., Tissuemizer)
  • Transfer pipet
  • Vortex
  • Large and small ultracentrifuge tubes (e.g., Beckman 38.5‐ml and 6.5‐ml polyallomer tubes)
  • Ultracentrifuge
  • Probe sonicator
  • Microplate protein assay kit (e.g., BioRad DC protein assay kit, BioRad)
  • Microplate reader
  • Heating block or boiling water bath
  • SDS‐acrylamide gel electrophoresis (SDS‐PAGE) equipment
  • Electrophoresis transfer equipment
  • Chemiluminescence‐detecting film (e.g., Hyperfilm ECL from Amersham)
  • Automatic film developer
  • Additional reagents and equipment for euthanasia of the mouse ( appendix 4H) and SDS‐PAGE (CPMB UNIT )

Support Protocol 1: Non‐Denaturing Protein Solubilization for Coimmunoprecipitation of Protein Complexes

  • Subcellular fractions of interest (from the protocol 1)
  • Deoxycholate (DOC) buffer (10% sodium deoxycholate in 500 mM Tris·Cl, pH 9.0)
  • Buffer T at 4°C (see recipe)
  • Dialysis buffer at 4°C (see recipe)
  • 36°C water bath
  • Dialysis cassettes (e.g., Slide‐A‐Lyzer Dialysis cassette, Pierce)
  • Magnetic stir bar
  • Stir plate

Support Protocol 2: Denaturing Protein Solubilization for Immunoprecipitation Studies of Protein Phosphorylation

  • Subcellular fraction samples of interest (from the protocol 1)
  • 20% sodium dodecyl sulfate (SDS) solution

Support Protocol 3: DSP‐Mediated Cross‐Linking of Proteins for Coimmunoprecipitation Studies

  • Frozen weight rat brain tissue (from the protocol 1)
  • 100 mM sodium borate buffer, pH 8.0, at 4°C
  • Dithiobis [succinimidylpropionate] (DSP)
  • Dimethylsulfoxide (DMSO)
  • 1 M Tris·Cl, pH 7.6 ( appendix 2A)
  • 15‐ml Falcon centrifuge tubes

Support Protocol 4: Immunoprecipitation of Phosphorylated Proteins and Coimmunoprecipitation of Protein Complexes

  Materials
  • PA or PG Sepharose beads (resuspended as a 20% slurry stock in 100 mM sodium borate buffer, pH 8.0, containing 0.02% sodium azide as a preservative)
  • 100 mM sodium borate buffer, pH 8.0, at 4°C
  • Immunoprecipitating antibody (for the detection of tyrosine phosphorylated proteins, the BD Biosciences monoclonal phosphotyrosine [PY20] antibody is recommended)
  • 20 mM dimethylpimelimidate dihydrochloride
  • 200 mM sodium borate buffer, pH 9.0, at 4°C
  • 200 mM ethanolamine, pH 8.0
  • Deionized water
  • Solubilized protein samples (e.g., from Support Protocols protocol 21 or protocol 32)
  • RIPA buffer at 4°C (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
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Figures

Videos

Literature Cited

Literature Cited
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