Protocol for in vitro BCR-mediated plasma cell differentiation and purification of chromatin-associated proteins

Autor: Marissa C. Franke, Evelyn Sievert, Kyoko Ochiai, Hiroki Shima, Tsuyoshi Ikura, Roger Sciammas, Kazuhiko Igarashi
Rok vydání: 2021
Předmět:
Science (General)
Cellular differentiation
1.1 Normal biological development and functioning
Cell
Plasma Cells
Immunology
Receptors
Antigen
B-Cell
Gene Expression
Mice
Transgenic
Plasma cell
General Biochemistry
Genetics and Molecular Biology
Transgenic
Mass Spectrometry
Q1-390
Mice
Tandem Mass Spectrometry
Underpinning research
Plasma cell differentiation
Receptors
Protocol
medicine
Animals
Flow Cytometry/Mass Cytometry
Receptor
Molecular Biology
Antibody
Chromatography
Liquid
General Immunology and Microbiology
Chemistry
General Neuroscience
breakpoint cluster region
B-Cell
Proteins
Cell Differentiation
Cell Biology
Chromatin immunoprecipitation
In vitro
Chromatin
Cell biology
Culture Media
medicine.anatomical_structure
Chromatin immunoprecipitation (ChIP)
Antigen
Generic health relevance
Chromatography
Liquid
Zdroj: STAR protocols, vol 2, iss 3
STAR Protocols, Vol 2, Iss 3, Pp 100633-(2021)
STAR Protocols
Popis: Summary Molecular-level understanding of plasma cell (PC) differentiation has been modeled using lipopolysaccharide (LPS) stimulation in vitro. However, this system does not involve the B-cell receptor (BCR)—a critical component of B cell biology. Here, we present a protocol for in vitro PC differentiation system dependent on BCR signaling that easily scales up for cell number-demanding applications, including protein complex purification. We describe how to set up this system and detail applications for endogenous complex purification of chromatin-associated proteins. For further details on the use and execution of this protocol, please refer to Sciammas et al. (2011) and Ochiai et al. (2018, 2020).
Graphical abstract
Highlights • In vitro plasma cell differentiation system via B-cell receptor signaling • Antibody conjugation of protein A or G beads for immunoprecipitation • Purification of endogenous protein complexes and chromatin-associated proteins • Gel extraction and digestion of proteins in preparation for LC-MS/MS analysis
Molecular-level understanding of plasma cell (PC) differentiation has been modeled using LPS stimulation in vitro. However, this system does not involve the B-cell receptor (BCR)—a critical component of B cell biology. Here, we present a protocol for in vitro PC differentiation system dependent on BCR signaling that easily scales up for cell number-demanding applications, including protein complex purification. We describe how to set up this system and detail applications for endogenous complex purification of chromatin-associated proteins.
Databáze: OpenAIRE
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