A Protein Cross‐Linking Assay for Measuring Cell Surface Expression of Glutamate Receptor Subunits in the Rodent Brain After In Vivo Treatments

Amy C. Boudreau1, Mike Milovanovic1, Kelly L. Conrad1, Christopher Nelson1, Carrie R. Ferrario1, Marina E. Wolf1

1 Department of Neuroscience, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.30
DOI:  10.1002/0471142301.ns0530s59
Online Posting Date:  April, 2012
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Abstract

Trafficking of neurotransmitter receptors between intracellular and cell surface compartments is important for regulating neurotransmission. We developed a method for determining if an in vivo treatment has altered receptor distribution in a particular region of rodent brain. After the treatment, brain slices are rapidly prepared from the region of interest. Then, cell surface–expressed proteins are covalently cross‐linked using the membrane‐impermeable, bifunctional cross‐linker bis(sulfosuccinimidyl)suberate (BS3). This increases the apparent molecular weight of surface receptors, while intracellular receptors are not modified. Thus, surface and intracellular receptor pools can be separated and quantified using SDS‐PAGE and immunoblotting. This method is particularly useful for analyzing AMPA receptor subunits, offering advantages in accuracy, efficiency, and cost compared to biotinylation. A disadvantage is that some antibodies no longer recognize their target protein after cross‐linking. We have used this method to quantify changes in receptor distribution after acute and chronic exposure to psychomotor stimulants. Curr. Protoc. Neurosci. 59:5.30.1‐5.30.19. © 2012 by John Wiley & Sons, Inc.

Keywords: BS3; cell surface expression; glutamate receptors; protein cross‐linking; receptor trafficking

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Cross‐Linking Surface‐Expressed Proteins in Freshly Dissected Brain Slices
  • Alternate Protocol 1: BS3 Cross‐Linking of Surface‐Expressed Proteins in Primary Neuronal Cultures
  • Support Protocol 1: Preparing the BS3 Stock Solution
  • Basic Protocol 2: SDS‐Page and Immunoblotting Analysis of Cross‐Linked Glutamate Receptor Subunits
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Cross‐Linking Surface‐Expressed Proteins in Freshly Dissected Brain Slices

  Materials
  • Lysis buffer (see recipe)
  • 1 M (10×) glycine stock solution in distilled H 2O
  • 52 mM (26×) BS3 stock solution in 5 mM sodium citrate, pH 5.0 (see protocol 3)
  • Artificial cerebrospinal fluid (aCSF), pH 7.4 (see recipe)
  • Live rats or mice
  • Brain matrix (we recommend a metal brain matrix, so that it can be chilled on ice, from ASI Instruments, https://asi‐instruments.com/)
  • Dissecting instruments including chilled tissue punch or scalpel
  • Glass petri dish
  • Whatman no. 1 filter paper (Whatman, cat. no. 1001‐070)
  • Guillotine
  • Double‐edged razor blades for use with brain matrix and tissue chopper
  • McIlwain‐type tissue chopper (Vibratome), set at 400 µm/slice
  • Metal spatula, chilled
  • Flat plate inverter (Thermo Scientific LABQUAKE Shaker, cat. no. C400110; used in rocking mode)
  • Laboratory timer
  • Refrigerated microcentrifuge capable of 20,000 × g
  • Sonicator (Sonic Dismembrator Model 100, Fisher Scientific)

Alternate Protocol 1: BS3 Cross‐Linking of Surface‐Expressed Proteins in Primary Neuronal Cultures

  • Primary rodent brain cell culture (see Chapter 3)
  • Hanks' balanced salt solution (HBSS; appendix 2A)
  • Cell scrapers

Support Protocol 1: Preparing the BS3 Stock Solution

  Materials
  • 5 mM sodium citrate buffer (prepared from 20× sodium citrate and citric acid stocks; see recipe in Reagents and Solutions
  • Bis(sulfosuccinimidyl)suberate (BS3; Pierce, cat. no. 21580; 50‐mg tubes; store at 4° to 8°C in a desiccator prior to use)

Basic Protocol 2: SDS‐Page and Immunoblotting Analysis of Cross‐Linked Glutamate Receptor Subunits

  Materials
  • Samples: lysates generated according to protocol 1 and removed from −80°C
  • BioRad DC Protein Assay kit
  • 2× sample treatment buffer [STB; see recipe, or purchase from BioRad (cat. no. 161‐0737)]
  • Tris⋅Cl polyacrylamide gradient (4%‐15%) gels (BioRad)
  • 1× electrophoresis running buffer (see recipe)
  • Precision Plus Prestained SDS‐PAGE standards (BioRad) or comparable visible molecular weight markers
  • MagicMark XP Western Standard (Invitrogen, cat. no. LC5602)
  • 1× transblotting buffer (see recipe)
  • 100% methanol
  • Tris buffered saline/Tween‐20 (TBS‐T; see recipe)
  • Blocking buffer (see recipe)
  • Primary antibodies (these are the antibodies that are currently able to recognize AMPA receptor subunits after they have been cross‐linked with BS3; however, each new lot of antibody should be tested with control cross‐linked tissue prior to use in experiments.):
    • Rabbit anti‐GluR1 (Thermo Scientific Pierce, cat. no. PA1‐37776)
    • Rabbit anti‐GluR2/3 (Chemicon, cat. no. AB1506)
    • Mouse anti‐GluR2 (Neuromab; Antibodies Inc., cat. no. 75‐002; http://www.antibodiesinc.com)
    • Rabbit anti‐GluR3 (Cell Signaling, cat. no. 3437)
  • Secondary antibodies:
    • Goat anti‐rabbit IgG‐HRP conjugate (Invitrogen, cat. no. G21234)
    • Goat anti‐Mouse IgG‐HRP conjugate (Invitrogen, cat. no. G21040)
  • ECL substrate (Amersham/GE Healthcare)
  • 0.1% Ponceau S (Sigma, cat. no. P7170‐1L; 1× stock)
  • Heat block
  • Mini‐PROTEAN 3 electrophoresis cell (BioRad, cat. no. 165‐3301)
  • BioRad power supply Power Pac HC (cat. no. 164‐5052)
  • Polyvinylidene fluoride (PVDF) membrane
  • Whatman 3MM chromatography paper
  • Plastic covered containers for incubations (PerfectWestern, http://genhunter.com)
  • Transblot sponges
  • Trans‐blot cell (BioRad, cat. no. 170‐3939)
  • Plate rocker (low to medium rocking)
  • Clear vinyl page protector sheets
  • Film (Amersham Hyperfilm ECL, cat. no. 28906839)
  • Autoradiography cassette (Fisher Scientific, cat. no. FBCA 810)
  • VersaDoc gel documentation system (BioRad; optional), or equivalent system
  • Total Lab (http://www.totallab.com/) or other comparable data analysis program
  • Gel scanner
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Figures

Videos

Literature Cited

Literature Cited
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