Regulatory Status Legend

Description

Component 51-6632AK consists of:

        51-6635KC        PBS Buffer (10X)                50 ml

        51-6636KC        Cell Lysis Buffer (1X)        50 ml

        51-6637KC        HEPES Buffer (2X)        50 ml

Component 51-6632BK consists of:

        51-66081U        Caspase-3 Fluorogenic Substrate                1 X 1.0 mg

                        N-acetyl-Asp-Glu-Val-Asp-AMC (7-amino-4-methylcoumarin)

        51-66221U        Caspase-3 Inhibitor                                        1 X 1.0 mg

                        Ac-DEVD-CHO [N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde)]

This kit is designed to measure caspase-3 or DEVD-cleaving activity, an early marker of cells undergoing apoptosis. The kit can also be used to determine if certain apoptosis pathways involve caspase-3 activation. It contains a caspase-3 fluorogenic substrate (Ac-DEVD-AMC), a caspase-3 aldehyde inhibitor (Ac-DEVD-CHO) and assay buffers.

Ac-DEVD-AMC is a synthetic tetrapeptide fluorogenic substrate (M.W. = 675 Daltons; purity ≥98%) that is used to identify and quantitate caspase-3 activity in apoptotic cell lysates. Ac-DEVD-AMC has been reported to have linear Michaelis-Menton kinetics with a Km of 10 µM for caspase-3. Caspase-3 cleaves the tetrapeptide between D and AMC, releasing the fluorescent AMC which can be quantified in cell lysates by ultraviolet (UV) spectrofluorometry using an excitation wavelength of 380 nm and an emission wavelength range of 420-460 nm. Apoptotic cell lysates containing active caspase-3 yield a considerable emission as compared to non-apoptotic cell lysates (Fig. 1).

Ac-DEVD-CHO is a potent, synthetic tetrapeptide inhibitor (M.W. = 502.5 Daltons; purity ≥98%) of caspase 3 activity. The inhibitor is used to block caspase-3 activity in apoptotic cells or cell lysates (Fig. 1). Ac-DEVD-CHO has been reported to have linear Michaelis-Menton kinetics with a Ki of less than 1 nM for Caspase-3. This Ki (1 nM) is significantly less than the Km of the Ac-DEVD-AMC substrate (10 µM). Thus, the amount of Ac-DEVD-CHO required for caspase-3 assays is much less than that of Ac-DEVD-AMC. When apoptotic cell lysates are incubated with both substrate and inhibitor, caspase-3 activity is blocked by Ac-DEVD-CHO. As a result, AMC is not released from Ac-DEVD-AMC and fluorescence is not emitted upon excitation of the samples.

Note: Investigators are advised that HEPES Buffer (2X) (Component 51-6637KC) can exhibit subtle color variation from clear to light yellow. This has been shown to not impact performance.

Preparation and Storage