Capture and Qualitative Analysis of the Activated Fc Receptor Complex from Live Cells

Angelica K. Florentinus1, Peter Bowden1, Veronica Barbisan1, John Marshall1

1 Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.22
DOI:  10.1002/0471140864.ps1922s67
Online Posting Date:  February, 2012
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This unit describes the isolation of activated Fc receptor complexes from RAW 264.7 macrophages using live‐cell affinity receptor chromatography (LARC). The Fc receptor complex is activated and captured by IgG‐coated microbeads on the surface of live macrophages. After the cells are disrupted, the receptor complexes are isolated by washing and sucrose gradient ultracentrifugation. Soluble proteins associated with the receptor complex are then eluted from the beads using a stepwise series of salt buffers and aqueous acetonitrile. The eluted proteins and the residual insoluble proteins on the beads can then be digested with trypsin and subjected to liquid chromatography, electrospray ionization, and tandem mass spectrometry (LC‐ESI‐MS/MS). Controls include IgG‐coated beads incubated with crude cell lysates or growth medium and beads coated with oxidized LDL or bovine serum albumin. Using this method, proteins present in IgG‐FcR complexes can be distinguished from those in control scavenger receptor complexes (oxLDL or BSA). Thus, LARC is capable of detecting specific members of IgG receptor supramolecular complexes.

Keywords: receptor complex; live cells; cognate ligand; ligand coated microbeads; live‐cell affinity receptor chromatography; LC‐ESI‐MS/MS; SQL

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Table of Contents

  • Introduction
  • Basic Protocol 1: Live‐Cell Affinity Receptor Chromatography (LARC): Capturing Activated Fc Receptor Complexes from the Surface of Live Raw 264.7 Macrophages
  • Support Protocol 1: Preparation of Control Microbeads
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Live‐Cell Affinity Receptor Chromatography (LARC): Capturing Activated Fc Receptor Complexes from the Surface of Live Raw 264.7 Macrophages

  • RAW 264.7 macrophages (ATCC no. TIB‐70)
  • DMEM or alpha MEM (Cellgro)
  • 5% fetal calf serum (FCS; PAA Laboratories)
  • 10% (v/v) suspension of 2‐µm polystyrene beads (∼2.185 × 1010 microbeads/ml; Bangs Laboratories Inc., cat. no. PS05N; store at 4°C)
  • 0.5 mg/ml human IgG (see recipe)
  • 1× PBS ( appendix 2E), 4°C
  • 60% (w/v) sucrose in 1× PBS (store up to 72 hr at 4°C)
  • Isotonic experimental medium (see recipe), 4°C
  • Homogenization buffer (see recipe), 4°C
  • Series of NaCl solutions (50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, and 1000 mM) in 1× PBS (store up to 1 month at 4°C)
  • 50% (v/v) acetonitrile in PBS made with HPLC‐grade water, 4°C
  • 2% (w/v) SDS ( appendix 2E)
  • Bovine serum albumin (BSA) standards
  • 5% and 50% (v/v) acetonitrile in 50 mM Tris‐HCl, pH 8.85, containing 200 mM urea
  • 1 µg/µl trypsin (Roche) in 1% (v/v) acetic acid (store up to 2 weeks at 4°C)
  • 80% (v/v) chloroform in 50 mM Tris, pH 8.5, made with HPLC‐grade water, 4°C
  • 10% (v/v) formic acid
  • Dithiothreitol (DTT)
  • Buffer A: 5% (v/v) acetonitrile, 5% (v/v) formic acid (store up to 1 month in a tightly sealed bottle)
  • Buffer B: 65% (v/v) acetonitrile, 5% (v/v) formic acid (store up to 1 month in a tightly sealed bottle)
  • 75‐cm2 culture flasks
  • Cell culture wheel rotator (“Ferris wheel”)
  • Ultracentrifuge with TLA110 rotor (or equivalent) and 1.7‐ml ultracentrifuge tubes
  • Cell scraper
  • 15‐ml Falcon tubes
  • French Press, precooled on ice
  • Light microscope and microscope slides
  • Microcentrifuge tubes
  • Syringe
  • Rotovap (Labconco)
  • C18 ZipTips (Millipore)
  • Electrospray mass spectrometer with:
    • 20‐µl sample loop
    • C18 LC‐MS column (300‐µm i.d., 15 cm; Vydek)
    • Agilent 1100 HPLC pump
    • Thermo LCQ ion trap
    • SEQUEST software
  • Additional reagents and equipment for dot blotting (e.g., unit 3.4) and SDS‐PAGE (units 10.1& 10.5)
NOTE: Prepare buffers, especially buffers that contain a carbon source such as sucrose, fresh the day before use and independently for each replicate of the experiment.
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Literature Cited

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