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.
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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
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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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Hou, T., Rinderknecht, C., Ghosh, D., Hadjinicolaou, A.V., Busch, R., Mellins, E.D. (2019). Pulse–Chase Analysis for Studies of MHC Class II Biosynthesis, Maturation, and Peptide Loading. In: van Endert, P. (eds) Antigen Processing. Methods in Molecular Biology, vol 1988. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9450-2_23
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