title: Measurement of Peptide Binding to MHC Class II Molecules by Fluorescence Polarization.
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.
authors.affiliation: Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts.
authors.forename: Liusong
authors.initials: L
authors.lastname: Yin
authors.affiliation: Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts.|Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Stern
mesh_terms: D000818:Animals; D017951:Antigen Presentation; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000276:immunology; D005454:Fluorescence Polarization / Q000379:methods; D000949:Histocompatibility Antigens Class II / Q000737:chemistry / Q000276:immunology; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000276:immunology
chemical_list: D018984:Epitopes, T-Lymphocyte; D000949:Histocompatibility Antigens Class II; D010455:Peptides
keywords: IC50; MHCII; binding competition; fluorescence polarization; peptide binding
references.citation: Cell. 1994 Jan 28;76(2):287-99
references.pmid: 8293464
references.citation: Immunogenetics. 2011 Jun;63(6):325-35
references.pmid: 21305276
references.citation: Curr Protoc Immunol. 2013 Feb;Chapter 18:Unit 18.3.
references.pmid: 23392640
references.citation: J Immunol. 2009 May 15;182(10):6342-52
references.pmid: 19414787
references.citation: Nat Chem Biol. 2006 Apr;2(4):197-201
references.pmid: 16505807
references.citation: Eur J Immunol. 2002 Mar;32(3):662-70
references.pmid: 11857340
references.citation: J Virol. 2012 May;86(9):4776-92
references.pmid: 22357271
references.citation: PLoS Biol. 2005 Mar;3(3):e91
references.pmid: 15760272
references.citation: Proc Natl Acad Sci U S A. 2012 Jul 10;109(28):11276-81
references.pmid: 22733780
references.citation: Biochem Pharmacol. 1973 Dec 1;22(23):3099-108
references.pmid: 4202581
references.citation: Proc Natl Acad Sci U S A. 2001 Oct 23;98(22):12450-5
references.pmid: 11606721
references.citation: Protein Expr Purif. 1999 Feb;15(1):105-14
references.pmid: 10024477
references.citation: Nat Immunol. 2007 Jan;8(1):92-100
references.pmid: 17143275
references.citation: J Immunol. 2010 Oct 1;185(7):4189-98
references.pmid: 20810981
references.citation: Nature. 1994 Mar 17;368(6468):215-21
references.pmid: 8145819
references.citation: J Immunol. 2010 Feb 1;184(3):1153-8
references.pmid: 20038641
references.citation: Proc Natl Acad Sci U S A. 2011 Nov 29;108(48):19329-34
references.pmid: 22084083
references.citation: Methods Mol Biol. 2013;960:447-459
references.pmid: 23329506
references.citation: J Immunol. 2004 Jan 15;172(2):1109-17
references.pmid: 14707085
references.citation: Immunology. 2008 Mar;123(3):305-13
references.pmid: 18251991
references.citation: Curr Protoc Immunol. 2001 May;Chapter 18:Unit 18.1
references.pmid: 18432744
references.citation: PLoS One. 2008;3(11):e3722
references.pmid: 19005572
references.citation: J Immunol Methods. 1993 Aug 9;163(2):209-16
references.pmid: 8354890
references.citation: Nature. 1995 Jun 29;375(6534):802-6
references.pmid: 7596415
references.citation: J Immunol. 2011 Sep 1;187(5):2442-52
references.pmid: 21775680
references.citation: J Immunol. 2006 Apr 1;176(7):4208-20
references.pmid: 16547258
references.citation: J Exp Med. 1992 Oct 1;176(4):1007-13
references.pmid: 1402647
references.citation: Nature. 1990 Jun 14;345(6276):615-8
references.pmid: 2190094
references.citation: Cell. 2012 Dec 21;151(7):1557-68
references.pmid: 23260142
references.citation: J Immunol. 1990 Mar 1;144(5):1849-56
references.pmid: 2307844
references.citation: Sci Rep. 2012;2:386
references.pmid: 22545194
references.citation: Cell. 1992 Feb 7;68(3):465-77
references.pmid: 1371238
references.citation: Nat Struct Mol Biol. 2013 Jan;20(1):90-8
references.pmid: 23222639
references.citation: J Immunol. 2012 Oct 15;189(8):3983-94
references.pmid: 22966084
references.citation: J Immunol. 1994 Jan 15;152(2):609-19
references.pmid: 8283041
references.citation: Biochemistry. 2000 Apr 4;39(13):3751-62
references.pmid: 10736175
references.citation: Biochemistry. 1999 May 4;38(18):5878-87
references.pmid: 10231540
references.citation: J Immunol. 2009 Oct 1;183(7):4187-91
references.pmid: 19767569
references.citation: Nat Immunol. 2011 Jan;12(1):54-61
references.pmid: 21131964
references.citation: Mol Immunol. 2009 Sep;46(15):3157-62
references.pmid: 19647320