Protocol

Antigen Processing

Volume 960 of the series Methods in Molecular Biology pp 411-432

Date:

Pulse–Chase Analysis for Studies of MHC Class II Biosynthesis, Maturation, and Peptide Loading

  • Tieying HouAffiliated withDepartment of Pediatrics, Division of Transplantation Biology and Immunology, Stanford University Medical School
  • , Cornelia H. RinderknechtAffiliated withDivision of Transplantation Biology and Immunology, Department of Pediatrics, Stanford University Medical School
  • , Andreas V. HadjinicolaouAffiliated withDivision of Transplantation Biology and Immunology, Department of Pediatrics, Stanford University Medical School
  • , Robert BuschAffiliated withDivision of Rheumatology, Department of Medicine, University of Cambridge, Addenbrooke’s Hospital
  • , Elizabeth MellinsAffiliated withDivision of Transplantation Biology and Immunology, Department of Pediatrics, Stanford University Medical School Email author 

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Abstract

Pulse–chase analysis is a commonly used technique for studying the synthesis, processing and transport of proteins. Cultured cells expressing proteins of interest are allowed to take up radioactively labeled amino acids for a brief interval (“pulse”), during which all newly synthesized proteins incorporate the label. The cells are then returned to nonradioactive culture medium for various times (“chase”), during which proteins may undergo conformational changes, trafficking, or degradation. Proteins of interest are isolated (usually by immunoprecipitation) and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the fate of radiolabeled molecules is examined by autoradiography. This chapter describes a pulse–chase protocol suitable for studies of major histocompatibility complex (MHC) class II biosynthesis and maturation. We discuss how results are affected by the recognition by certain anti-class II antibodies of distinct class II conformations associated with particular biosynthetic states. Our protocol can be adapted to follow the fate of many other endogenously synthesized proteins, including viral or transfected gene products, in cultured cells.

Key words

MHC class II Biosynthesis Maturation Metabolic labeling Immunoprecipitation