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Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3
Published on: December 20, 2013
Zebrafish caspase-3: molecular cloning, characterization, crystallization and phylogenetic analysis
Chiranjib Chakraborty1, Shyam Sundar Nandi, Surajit Sinha
1Department of Biotechnology, College of Engineering and Technology, IILM Academy of Higher Learning, Knowledge Park-II, Greater Noida, (UP), India. drchiranjib@yahoo.com
Protein and Peptide Letters
|July 18, 2006
Summary
Zebrafish caspase-3, crucial for apoptosis, was cloned and characterized. Overexpression induced apoptotic morphology in fish kidney cells, confirming its biological activity and structural properties.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Apoptosis is vital for tissue homeostasis across species.
- Caspase-3 is a key executioner caspase in vertebrate apoptosis.
Purpose of the Study:
- To clone and characterize zebrafish caspase-3.
- To investigate its role in apoptosis and determine its structural properties.
Main Methods:
- Zebrafish caspase-3 was cloned and expressed in E. coli.
- Western blotting and transfection assays were used to confirm expression and activity.
- X-ray crystallography was employed for structural determination.
Main Results:
- Western blotting identified precursor and mature forms of caspase-3.
- Transfection of fish kidney cells with zebrafish caspase-3 induced apoptotic morphology.
- Phylogenetic analysis revealed ~60% identity with other vertebrate caspase-3s.
- Orthorhombic crystals of caspase-3 were obtained, enabling X-ray diffraction.
Conclusions:
- Zebrafish caspase-3 is a functional homolog of caspase-3 in other vertebrates.
- Its expression triggers apoptosis, and its structure has been elucidated through crystallography.
