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Purification and use of granzyme B
1Manitoba Institute of Cell Biology, University of Manitoba, Winnepeg, Canada.
Methods in Enzymology
|July 29, 2000
Summary
Granzyme B (GrB) induces apoptosis in target cells, a process crucial for cytotoxic T lymphocyte (CTL) and natural killer (NK) cell function. Methods are detailed for using GrB, perforin, and adenovirus to trigger and measure apoptosis.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Granzyme B (GrB) is a key protease released by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells.
- GrB mediates apoptosis (programmed cell death) in target cells, often in conjunction with perforin.
- Adenovirus can also induce GrB-mediated apoptosis, serving as a perforin alternative.
Purpose of the Study:
- To describe methods for purifying and quantifying Granzyme B and perforin.
- To detail the preparation and titration of adenovirus for apoptosis induction.
- To outline assays for detecting GrB apoptotic activity in tumor cells.
Main Methods:
- Purification and quantitation of Granzyme B and perforin.
- Preparation and titration of adenovirus.
- Application of GrB, perforin, and adenovirus to induce apoptosis in tumor cells.
- Assays for detecting GrB-mediated apoptosis.
Main Results:
- Established protocols for GrB and perforin purification and quantitation.
- Developed methods for adenovirus preparation and titration.
- Demonstrated the induction of apoptosis in tumor cells using these reagents.
- Validated assays for measuring GrB apoptotic activity.
Conclusions:
- The study provides comprehensive methods for utilizing Granzyme B, perforin, and adenovirus in apoptosis research.
- These methods facilitate the study of GrB's role in cell-mediated cytotoxicity and apoptosis.
- The described techniques are valuable for investigating apoptosis induction in cancer therapy research.