| Field | Specification |
|---|---|
| Assay Type | |
| Detection Instrument(s) | HTRF®-certified Microplate Reader (e.g. Tecan M1000, Tecan Spark) |
| Detection Method | |
| Product Type | |
| Shipping | |
| Storage |
Background
Caspase-3 is a critical enzyme involved in the process of apoptosis, or programmed cell death. It’s considered one of the “effector caspases,” which are the final executioners of the apoptotic process. Caspase-3 is activated in response to various cellular stress signals and, once activated, it leads to the breakdown of cellular components and the death of the cell. This ensures that damaged or unwanted cells are efficiently removed without causing harm to the surrounding tissue. In the context of cancer, the role of caspase-3 is significant because cancer cells often evade apoptosis, allowing them to survive and proliferate uncontrollably. The dysregulation of apoptosis is a hallmark of cancer, and as a result, many cancer cells have reduced or altered caspase-3 activity. Because of its essential role in apoptosis, caspase-3 has been studied in the context of cancer (where apoptosis may be disrupted) and neurodegenerative diseases (where increased apoptosis can occur).
Assay Principle
Caspase-3 activity assay kit is designed to measure caspase-3 activity for enzyme profiling and inhibitor screening . Proteolytic activity of caspase-3 cuts the fluorogenic substrate and releases the fluorophore, resulting in fluorescent intensity increase which can be measured using a microplate reader at excitation at 360 nm and emission at 460 nm.
Application
Quantification of caspase-3 activity and High throughput screening of compounds that have effects on the enzyme activity for drug discovery.
Instrument Required
A microplate reader capable of measuring fluorescence intensity at excitation at 360 nm and emission at 460 nm.
Kit Components
| Catalog No. | Item | Amount | Storage |
|---|---|---|---|
| 2x Caspase assay buffer | 25 mL | -20°C | |
| 384-well microplate, White | Room temperature | ||
| Materials needed but not supplied |
Materials Not Supplied
- Microplate reader
- 0.5 M DTT
- Adjustable micro-pipettor
- Sterile Tips
Assay Protocol
- Step 1. Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare a series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).
- Step 2. Prepare caspase-3 solution Thaw caspase-3 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: caspase-3 protein is sensitive to freeze/thaw cycles. Limit the number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the caspase-3 protein 500-fold (1 µL caspase-3 + 499 µL 1X DTT-containing assay buffer). Add 8 µl of diluted protein solution to each positive control wells and inhibitor test wells. Add 8 µl of 1X DTT containing buffer to each of the negative control wells.
- Step 3. Add inhibitor solution Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each negative and positive control wells. Incubate at room temperature for 30 minutes (optional).
- Step 4. Prepare caspase-3 substrate Thaw the substrate at room temperature. Dilute the substrate 50-fold (1 µL of 1 mM tracer + 49 µL 1X DTT-containing assay buffer). Add 10 µl of diluted substrate to each well. Dilute enough substrate for single use. Store remaining undiluted tracer at -80°C. Do not re-use the diluted tracer.
- Step 5. Incubate the reaction at room temperature for 60 minutes.
- Step 6. Measure fluorescent intensity Fluorescent intensity should be measured by excitation wavelength at 360 nm and emission at 460 nm.
Data Analysis
% Activity = (S − N) / (P − N) × 100S = sample signal | P = positive control (100%) | N = negative control (0%)
Calculate percentage activity In the absence of the compound (positive control), the sample signal (P) is defined as 100% activity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% activity. The percent activity in the presence of each compound is calculated according to the following equation: % activity = (S-N)/(P-N) X100, where S= the sample signal in the presence of the compound.
Assay Validation
Assay: Caspase3 Activity
Validated IC50: 0.0006 uM
Biological Pathway / Process
Apoptosis (Caspase-3 execution pathway)
Therapeutic / Disease Area
Oncology; Neurodegenerative Disease
▶▼What activity does the Caspase-3 Activity Assay Kit measure?
This kit measures Caspase-3 (executioner caspase in apoptosis) enzymatic activity using a fluorescence-based format. The assay is designed for cell-free, homogeneous conditions that support both endpoint and kinetic measurements, and is suitable for IC₅₀ determination of inhibitor candidates.
▶▼What instrument or plate reader is required?
A fluorescence microplate reader is required to read this assay. The specific excitation and emission wavelengths depend on the detection mode used. Please refer to the product datasheet for exact instrument settings and compatible reader models.
▶▼How many reactions are included, and is bulk ordering available?
This kit is available in 384 reactions. Each reaction is conducted in a 96-well / 384-well format, making it directly compatible with standard liquid-handling robotics for HTS applications. For bulk orders or custom quantities, please contact BioHippo or submit a quote request — distributor pricing is available.
▶▼Has the assay performance been validated?
Yes. The assay has been validated with a reference inhibitor demonstrating an IC₅₀ of 0.0006 uM, confirming the assay window and signal-to-background ratio are suitable for inhibitor screening. The Z′ factor should be determined in your laboratory under your specific conditions.
▶▼What are the storage and shipping requirements?
The kit ships on Dry Ice and should be stored at -80°C upon receipt. Individual components may have different storage requirements — please refer to the component table in the datasheet. Protein components are sensitive to freeze–thaw cycles; aliquot on first thaw and avoid repeated freeze–thaw.
Need a different assay kit format or extra support beyond the catalog item? We offer customization and add-on services for assay kits to better fit your target, workflow, and detection platform. Options may include assay format customization (biochemical, binding, or cell-based), detection method selection such as TR-FRET, fluorescence polarization, luminescence, or colorimetric readout, target-specific reagent development including recombinant proteins, conjugates, antibodies, substrates, tracers, and controls, as well as protocol optimization for sensitivity, specificity, signal window, and reproducibility. We can also support kit component adjustments, plate format and scale options, QC and validation packages, bulk or custom packaging, and related assay development services when a standard kit does not fully meet your needs. Click Talk to a Scientist to submit a request, email us at support@biohippo.com, or explore our Research Services for additional support. Our team will be in contact with you shortly.
Research budgets are tight — we get it. That's why we've put together a fresh round of exclusive promotions designed to help you stock up on the reagents, kits, and consumables your lab depends on, without stretching your budget.
🔬 What's on offer right now:
10% Off Pre-Designed siRNA Sets — use code SIRNA10
20% Off Transmembrane Proteins — use code TM20
$50 Off All ELISA Kits — auto-applied at checkout (code ELISA50)
50% Off Lab Consumables + Free Shipping on orders $500+
15% Off Proteins from Trusted Suppliers — use code PROTEIN15
$99 Pipette Filler Promotion Package (reg. $236)
Save 10% on your first order of any R&D grade DENARASE® with code DENARASE10 at checkout.
