Ab102491 Detection Kit Caspase 3 Inhibitor Drug - Abcam

ab102491 Caspase 3 Inhibitor Drug Detection Kit

Instructions for Use

For the rapid, sensitive and accurate detection of Caspase 3 inhibition by various compounds This product is for research use only and is not intended for diagnostic use.

Version 3 Last updated 28 July 2015

Table of Contents

1. Overview

2

2. Protocol Summary

2

3. Components and Storage

3

4. Assay Protocol

5

5. Factors to consider for caspase activity assays

7

6. Troubleshooting

9

1. Overview

Caspases have been shown to play a crucial role in apoptosis induced by various deleterious and physiologic stimuli. Inhibition of caspases can delay apoptosis, implicating a potential role in drug screening efforts.

Abcam's Caspase 3 Inhibitor Drug Detection Kit provides an effective means for screening caspase inhibitors using fluorometric methods. The assay utilizes synthetic peptide substrate DEVD-AFC (AFC, 7-amino-4-trifluoromethyl coumarin). Active caspase 3 cleaves the synthetic substrate to release free AFC, which can then be quantified by fluorometry. Compounds to be screened can directly be added to the reaction and the level of inhibition of caspase 3 activity can be determined by comparison of the fluorescence intensity in samples with and without the testing inhibitors.

2. Protocol Summary

Sample Preparation

Prepare and Add Master Mix

Measure Fluorescence

3. Components and Storage

A. Kit Components Item

Quantity

2X Reaction Buffer Caspase Substrate DEVD-AFC (1 mM) DTT (1 M) Active Caspase 3 (Lyophilized) Caspase Inhibitor, Z-VAD-FMK (2 mM)

10 mL 0.5 mL 100 ?L 100 units

10 ?L

* Store kit at -20?C. All components are stable for 6 months under proper storage conditions.

Protect DEVD-AFC from light.

REACTION BUFFER: After thawing, store the 2X Reaction Buffer at +4?C. Aliquot enough 2X Reaction Buffer for the number of assays to be performed. Add DTT to the 2X Reaction Buffer immediately before use (10 mM final concentration: add 10 l of 1.0 M DTT stock per 1 ml of 2X Reaction Buffer).

ACTIVE CASPASE 3: Reconstitute in 550 l 2X Reaction Buffer. Aliquot and immediately store at -80?C.

B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Fluorescent microplate reader 96-well plate Orbital shaker

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