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

  • Tieying Hou
  • Cornelia Rinderknecht
  • Debopam Ghosh
  • Andreas V. Hadjinicolaou
  • Robert BuschEmail author
  • Elizabeth D. MellinsEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1988)

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 

Abbreviations

Abs

Antibodies

APCs

Antigen presenting cells

B-LCL

EBV-transformed B-lymphoblastoid cell lines

CLIP

Class-II-associated invariant chain peptides

Cys/Met

Cysteine/methionine

ER

Endoplasmic reticulum

Ii

Invariant chain

IP

Immunoprecipitation

LIP

Leupeptin-induced polypeptides

MHC

Major histocompatibility complex

MIIC

MHC class II compartments

PAS

Protein A Sepharose

PGS

Protein G Sepharose

PMSF

Phenylmethylsulfonyl fluoride

SDS-PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SLB

Standard Laemmli sample loading buffer

Notes

Acknowledgments

The initial work was supported by NIH grants F32 AI089090 to T.H. and AI095813 and AI28809 to E.D.M. R.B. was supported by a Senior Research Fellowship from Arthritis Research UK [ref. 18543]. D.G. is supported by a grant to E.D.M. from Codexis, Inc.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Tieying Hou
    • 1
    • 2
  • Cornelia Rinderknecht
    • 1
    • 3
  • Debopam Ghosh
    • 1
  • Andreas V. Hadjinicolaou
    • 1
    • 4
  • Robert Busch
    • 5
    Email author
  • Elizabeth D. Mellins
    • 1
    Email author
  1. 1.Program in Immunology, Department of PediatricsStanford University Medical SchoolStanfordUSA
  2. 2.Department of PathologyMD Anderson Cancer CenterHoustonUSA
  3. 3.Department of Translational ImmunologyGenentechSouth San FranciscoUSA
  4. 4.MRC Human Immunology Unit, Weatherall Institute of Molecular MedicineUniversity of OxfordOxfordUK
  5. 5.Department of Life SciencesUniversity of RoehamptonLondonUK

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