Detection of Caspase-3 activity in apoptotic cells

Summary

The detection of Caspase-3 activity in apoptosis is mainly applied to (1) enzyme viability assay (2) apoptosis index assay.

Operation method

Detection of Caspase-3 Activity

Principle

The caspase family plays a very important role in mediating apoptosis, with caspase-3 as a key executive molecule that functions in many pathways of apoptotic signaling. caspase-3 normally exists in the cytoplasm as an zymogen (32KD), and is activated during the early stages of apoptosis, with the activated caspase-3 comprising two large subunits (17KD) and two small subunits (12KD), which cleave the corresponding cytoplasmic cytosolic substrates, eventually leading to apoptosis. However, in the late stage of apoptosis and dead cells, the activity of caspase-3 decreases significantly.

Materials and Instruments

Cells
Phosphate buffer Distilled water Skimmed milk powder Caspase-3 poly antibody Tween Ac-DEVD-AMC
Nitrocellulose membrane PVDF membrane Fluorescence spectrophotometer UV flow cytometer Beakers Pipettes Drainage sticks

Move

1, Collection of cells;

2, PBS washing;

3, Extraction of cell lysate;

4, Protein quantification;

5, SDS-PAGE electrophoresis, nitrocellulose membrane or PVDF membrane transfer, 5% skimmed milk powder closed, 1.5--2h at room temperature or overnight at 4°C;

6, Caspase-3 poly or monoclonal antibody reaction at room temperature 1--2h or 4°C overnight, TBS-T (TBS containing 0.05% Tween 20) washed 3 times, 5--10min/time;

7、HRP-labeled sheep anti-mouse IgG or AP-labeled sheep anti-mouse IgG reacted at room temperature for 1--2h→ TBS-T washed 3 times, 5--10min/time;

8, ECL development or NBT/BCIP color development.

Caveat

1. Be as gentle as possible throughout the operation, do not blow the cells with force;2. test within one hour as soon as possible after the reaction is completed.

Common Problems

1. The results were analyzed by Western blot for activation of Procaspase-3, and cleavage of activated Caspase-3 and to the substrate poly(ADP-ribose) polymerase [poly(ADP-ribose) polymerase, PARP], etc.

2. Fluorescence spectrophotometry and flow cytometry analysis are also available.

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