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Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method
Published on: October 23, 2018
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Protocol for visualizing newly synthesized proteins in primary mouse hepatocytes
Yuqian Shen1,2, Wenhua Liu1, Jian Zuo1
1School of Life Science and Technology, the Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing 210096, China.
STAR Protocols
|June 30, 2021
Summary
This study introduces a method to track newly synthesized proteins in mouse cells using a special amino acid and click chemistry. This allows researchers to visualize and localize newly made proteins accurately.
Area of Science:
- Biochemistry
- Cell Biology
- Proteomics
Background:
- Distinguishing newly synthesized proteins from existing ones is difficult due to a shared amino acid pool.
- L-homopropargylglycine, a methionine analog with an alkyne group, can be incorporated into nascent proteins.
- This alkyne moiety enables click chemistry reactions for protein labeling.
Purpose of the Study:
- To develop and present an integrated tool for the selective identification and localization of newly synthesized proteins.
- To provide a reliable method for tracing protein synthesis in isolated primary mouse hepatocytes.
Main Methods:
- Utilizing L-homopropargylglycine, an amino acid analog of methionine containing an alkyne group.
- Employing click chemistry to react the alkyne moiety with azide-containing Alexa Fluor.
- Implementing immunofluorescence staining to visualize and localize the labeled nascent proteins.
Main Results:
- Demonstration of an effective method for tracing and localizing newly synthesized proteins.
- Successful application of the integrated tool in isolated primary mouse hepatocytes.
- Accurate visualization of nascent protein synthesis through immunofluorescence staining.
Conclusions:
- The presented integrated tool enables the selective identification and localization of newly synthesized proteins.
- This method provides a valuable approach for studying protein synthesis dynamics in cellular contexts.
- The protocol offers a reliable way to trace nascent proteins in biological research.
