Binding of biotinylated peptides to MHC class II proteins on cell surfaces.
This unit describes a simple, reproducible, and semiquantitative assay for measuring ligand binding to cell surface receptors. This approach is useful as a quick screen for peptide binding to MHC class II proteins that avoids the need to purify the MHC class II molecules and that uses small numbers of cells. The basic protocol describes procedures for incubating cells expressing the MHC molecule of interest with a biotinylated peptide, washing off excess peptide, and detecting the bound peptides by staining with fluorescently labeled avidin. Bound fluorescence is then quantitated by flow cytometry. An alternate protocol provides a more sensitive method of measuring the peptide-receptor complexes, and is useful when receptor density or binding site availability become limiting factors in the basic protocol. The alternate protocol uses an avidin/anti-avidin/avidin sandwich, which has been found to increase sensitivity substantially without sacrificing specificity. Support protocols are provided for biotinylation of synthetic peptides in solution and for preparation of peptide stock solutions for use in the binding assays.
Rothbard
Jonathan B
JB
Stanford University Medical Center, Stanford, California.
Busch
Robert
R
Stanford University Medical Center, Stanford, California.
D000938:Antigen-Presenting Cells / Q000737:chemistry / Q000378:metabolism; D001360:Avidin / Q000737:chemistry* / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D019921:Biotinylation; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006684:HLA-DR Antigens / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding
D006684:HLA-DR Antigens; D010455:Peptides; D001360:Avidin; D001710:Biotin