Caspase-3 Fluorometric Assay Kit
This Caspase-3 Fluorometric Assay Kit is designed to detect caspase-3(an early marker of cells undergoing apoptosis) or DEVD-cleaving activity, and determine if certain apoptosis pathways involve caspase-3 activation. The kit contains a caspase-3 fluorogenic substrate (Ac-DEVD-AMC), a caspase-3 aldehyde inhibitor (Ac-DEVD-CHO) and assay buffers. Ac-DEVD-AMC, a synthetic tetrapeptide fluorogenic substrate, is used to identify and quantitate caspase-3 activity in apoptotic cell lysates, and 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. Ac-DEVD-CHO is a potent, synthetic tetrapeptide inhibitor of caspase 3 activity and is used to block caspase-3 activity in apoptotic cells or cell lysates 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.
PBS Buffer (10X): 30 ml
Cell Lysis Buffer (1X): 30 ml
Reaction Buffer (2X): 10 ml
N-acetyl-Asp-Glu-Val-Asp-AMC (7-amino-4-methylcoumarin): 1.0 mg
Ac-DEVD-CHO [N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde)]: 1.0 mg
Store at ≤ -20 °C for up to 6 months after initial use. Avoid repeated freeze-thaw cycles.
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