Measurement of Peptide Binding to MHC Class II Molecules by Fluorescence Polarization

Liusong Yin1, Lawrence J. Stern2

1 Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts, 2 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 5.10
DOI:  10.1002/0471142735.im0510s106
Online Posting Date:  August, 2014
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Abstract

Peptide binding to major histocompatibility complex class II (MHCII) molecules is a key process in antigen presentation and CD4+ T cell epitope selection. This unit describes a fairly simple but powerful fluorescence polarization‐based binding competition assay to measure peptide binding to soluble recombinant MHCII molecules. The binding of a peptide of interest to MHCII molecules is assessed based on its ability to inhibit the binding of a fluorescence‐labeled probe peptide, with the strength of binding characterized as IC50 (concentration required for 50% inhibition of probe peptide binding). Data analysis related to this method is discussed. In addition, this unit includes a support protocol for fluorescence labeling peptide using an amine‐reactive probe. The advantage of this protocol is that it allows simple, fast, and high‐throughput measurements of binding for a large set of peptides to MHCII molecules. Curr. Protoc. Immunol. 106:5.10.1‐5.10.12. © 2014 by John Wiley & Sons, Inc.

Keywords: MHCII; peptide binding; fluorescence polarization; binding competition; IC50

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Peptide Binding to MHCII Molecules as IC50 Using a Fluorescence Polarization‐Based Binding Competition Assay
  • Support Protocol 1: Labeling of the Probe Peptide with a Fluorochrome
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Peptide Binding to MHCII Molecules as IC50 Using a Fluorescence Polarization‐Based Binding Competition Assay

  Materials
  • Alexa488 labeled influenza hemagglutinin‐derived probe peptide (Alexa488‐FRR‐HA 306‐318; see protocol 2Support Protocol for the labeling)
  • Double‐distilled water (ddH 2O; Millipore)
  • Unlabeled target peptides (21st Century Biochemicals)
  • Dimethyl sulfoxide (DMSO)
  • Purified MHCII molecules (HLA‐DR1 in this protocol) (see unit 18.3)
  • Binding buffer, pH 5.5 (see recipe)
  • Protease inhibitor cocktail (prepare fresh; see recipe)
  • Phosphate‐buffered saline (PBS), pH 7.4 (see recipe)
  • 1.5‐ml black microtubes
  • 1.5‐ml clear microtubes
  • 96‐well polypropylene flat‐bottom nonbinding black microplate (Greiner)
  • Aluminum Sealing Foil for 96‐well Microplate (USA Scientific)
  • 37°C incubator
  • VICTOR X5 Multilabel Plate Reader (PerkinElmer), POLARstar OPTIMA Plate Reader (BMG LABTECH) or other fluorescence microplate reader equipped for fluorescence polarization
  • GraphPad Prism 6 graphing and data analyzing software

Support Protocol 1: Labeling of the Probe Peptide with a Fluorochrome

  Materials
  • Influenza hemagglutinin (HA) analog peptide FRR‐HA 306‐318 (Ac‐PRFVKQNTLRLAT; 21st Century Biochemicals)
  • 150 mM sodium bicarbonate buffer, pH 9.0 (or other non‐amine buffer) (made fresh)
  • Alexa Fluor 488 Carboxylic Acid, 2,3,5,6‐Tetrafluorophenyl Ester (Alexa488‐TFP; Invitrogen)
  • Buffer A [5% triflouroacetic acid (TFA) in ddH 2O)]
  • Buffer B (5% TFA in acetonitrile)
  • 1.5‐ml black microtubes
  • Jupiter 5u C18 300A 250 × 4.6 mm column (C18 column; Phenomenex)
  • Agilent 1200 series Liquid Chromatography system (LC; Agilent Technologies)
  • Lyophilizer
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Figures

Videos

Literature Cited

Literature Cited
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