Chemical cross-linking to study protein self-assembly in cellulo

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Proteins frequently self-assemble into dimers and higher-order oligomers.
• Understanding these assemblies is crucial for deciphering protein function and cellular processes.
• Characterizing conformational states of endogenous proteins provides insights into their biological roles.

Purpose of the Study:

• To present a detailed protocol for assessing protein self-assembly in cell lysates.
• To enable the characterization of conformational states of endogenous proteins of interest.
• To provide a reliable method for studying protein oligomerization in a cellular context.

Main Methods:

• Chemical cross-linking of proteins in cell lysates.
• Cell culture and preparation of cell lysates.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
• Western blotting for endogenous protein detection.

Main Results:

• The protocol effectively assesses protein self-assembly in cell lysates.
• It allows for the characterization of conformational states of endogenous proteins.
• Demonstrated applicability using recombinant proteins and endogenous targets.

Conclusions:

• Chemical cross-linking is a viable method for studying protein self-assembly in cell lysates.
• This protocol facilitates the investigation of endogenous protein conformational states.
• The method aids in understanding protein oligomerization and interactions within cells.