Are the Caspase Activity Assays specific for the only Caspase indicated on the insert?

The Caspase Activity Assays contain the substrate preferred by the individual Caspase being assayed. However, none of these substrates is completely specific for one Caspase. If more specificity is required, consider an ELISA which uses a specific capture antibody.

Why are there two different types of TUNEL detection kits, TdT and TACS-XL, and which should I use?

There are two different TUNEL (terminal dUTP Nicked End Labeling) methods for apoptosis detection. The first uses TdT (Terminal deoxynucleotidyl Transferase) to incorporate biotinylated nucleotides into the 3'-OH ends of the DNA fragments. This labeling is optimized by the addition of a cation. The biotinylated nucleotides are then detected by using a streptavidin-HRP-conjugate followed by the substrate. The TACS-XL kits also use TdT to incorporate nucleotides into the 3'-OH ends of the DNA fragments. However, these nucleotides are BrdU-labeled rather than directly biotinylated. A biotinylated anti-BrdU antibody is then used for detection. More efficient incorporation of BrdUTP may result in improved signal-to-noise ratio.

Are the Caspase Activity Assays quantitative?

The Caspase Activity Assays are considered semi-quantitative. The results are best used to determine a fold increase in the Caspase activity in apoptotic cells over that of non-induced cells. The use of a background control (reactions where no cell lysate or substrate is added) is preferred. If this control gives a substantial reading, it should be subtracted from the experimental results prior to calculating the fold increase.

Does R&D Systems offer a positive control for the Caspase Activity Assays?

R&D Systems offers various recombinant Caspase enzymes that are ideal for use as positive controls. If one prefers to generate a positive control, there are several published methods. These include incubating cells with 2 µM Staurosporin for 2 hours, which induces apoptosis in most cell types. R&D Systems Technical Service has a list of references for generating positive controls for specific Caspases.

How are the Caspase Activity Assays used?

Caspase Activity Assays provide a simple and convenient means for assaying protease activity in cell lysates. Upon cleavage of the Caspase-specific peptide substrate, a free reporter molecule can be quantified using a colorimetric or fluorometric microtiter plate reader. Comparison of the signal generated from an apoptotic sample with an uninduced control allows determination of the fold increase in Caspase activity. The level of Caspase activity is directly proportional to the signal detected.

How are the Caspase Activity Assays different from the Caspase ELISA Kits?

The Caspase ELISA Kits measure the total amount of Caspase present in the sample, regardless of the level of activity of the Caspase. These kits are more specific than the activity assays due to the use of antibodies for caspase recognition. The Capase Activity Assays offer a qualitative comparison of the Caspase activity in different samples, regardless of the total amount of Caspase. Specificity of Caspase enzymes are not absolute rather they are relatively specific. Activity assays utilize the cleavage of a peptide as a measure of activity. Although a Caspase may have a preference for a particular peptide sequence, it may cleave similar sequences under the right conditions or given enough time.

Are R&D Systems Caspase Inhibitors irreversible?

Yes, the majority of R&D Systems Caspase Inhibitors have a Fluoromethyl ketone (FMK) functional group on the C-terminus of the peptide, and act as effective irreversible inhibitors with no added cytotoxic effects. Inhibitors synthesized with a benzyloxycarbonyl group (also known as BOC or Z) at the N-terminus and O-methyl side chains exhibit enhanced cellular permeability. R&D Systems also offers a General Caspase Inhibitor, Q-VD-OPh, Catalog # OPH001, as well as a FITC-conjugated pan-caspase inhibitor (ApoStat), Catalog # FMK012, which are both cell-permeable, irreversible inhibitors of caspase activity.

Caspase Inhibitor Control Z-FA-FMK

Name: Caspase Inhibitor Control Z-FA-FMK
Brand: R&D Systems
SKU: FMKC01

R&D Systems, part of Bio-Techne | Catalog # FMKC01

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Key Product Details

Cell permeable fluoromethyl ketone (FMK)-derivatized peptides act as effective irreversible Caspase inhibitors with no cytotoxic effects and, therefore, are useful tools for studying Caspase activity.

Conjugate

Unconjugated

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Formulation, Preparation and Storage

FMKC01

Shipping	The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage	Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.

Background: Caspase (Pan-specific)

Caspases are a family of cytosolic aspartate-specific cysteine proteases involved in the initiation and execution of apoptosis. They are expressed as latent zymogens and are activated by an autoproteolytic mechanism or by processing by other proteases (frequently other caspases). Human caspases can be subdivided into three functional groups: cytokine activation (caspase-1, -4, -5, and -13), apoptosis initiation (caspase-2, -8, -9, -and -10), and apoptosis execution (caspase-3, -6, and -7).

Caspases are regulated by a variety of stimili, including APAF1, CFLAR/FLIP, NOL3/ARC, and members of the inhibitor of apoptosis (IAP) family such as BIRC1/NAIP, BIRC2/cIAP-1, BIRC3/cIAP-2, BIRC4/XIAP, BIRC5/Survivin, and BIRC7/Livin. IAP activity is modulated by DIABLO/SMAC or PRSS25/HTRA2/Omi. Cell-permeable and irreversible peptide inhibitors are also available for different caspases.

Alternate Names

CASP1, caspase 1, ICE, IL1BC, p45

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Product Specific Notices for Caspase Inhibitor Control Z-FA-FMK

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