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A rapid assay for detecting cellular TdT enzymatic activity
W Harriman1, E R Fischer, M Wabl
1Department of Microbiology and Immunology, University of California, San Francisco 94143-0670, USA.
Journal of Immunological Methods
|April 26, 1995
Summary
A new solid-phase assay quantifies terminal deoxynucleotidyl transferase (TdT) enzymatic activity in cell extracts. This simple, reproducible method is ideal for high-throughput screening of TdT activity in limited cell samples.
Area of Science:
- Biochemistry
- Molecular Biology
- Assay Development
Background:
- Terminal deoxynucleotidyl transferase (TdT) is a key enzyme in DNA metabolism.
- Quantifying TdT enzymatic activity is crucial for various research applications.
- Existing methods may be complex or require large sample volumes.
Purpose of the Study:
- To develop a novel, simplified solid-phase assay for TdT enzymatic activity.
- To enable accurate quantification of TdT in crude cellular extracts.
- To facilitate high-throughput analysis of TdT activity.
Main Methods:
- Development of a solid-phase assay utilizing microtiter plates.
- Immobilization of affinity-purified, polyclonal anti-TdT antibodies onto the plate wells.
- Direct measurement of TdT enzymatic activity bound to immobilized antibodies in cellular extracts.
Main Results:
- The assay provides highly reproducible quantification of TdT enzymatic activity.
- The method is effective even for samples with low TdT activity.
- The assay is technically simple and suitable for large-scale screening.
Conclusions:
- A robust and user-friendly solid-phase assay for TdT activity has been established.
- This assay is well-suited for analyzing numerous clones with limited cellular material.
- The developed method simplifies the assessment of TdT enzymatic function in biological samples.