Caspase-3/CASP3 Protein, Human

Caspase-3/CASP3 protein is a thiol protease critical in apoptosis and is activated by initiating caspases (CASP8, CASP9 and/or CASP10). It catalyzes the cleavage of multiple proteins in sympathetic neurons, including PARP1, SREBP, caspase-6, -7 and -9, huntingtin, and RET. Caspase-3/CASP3 Protein, Human is the recombinant human-derived Caspase-3/CASP3 protein, expressed by E. coli, with tag free.

Caspase-3/CASP3 Protein functions as a pivotal thiol protease in the execution phase of apoptosis, serving as a major effector caspase. Upon cleavage and activation by initiator caspases (CASP8, CASP9, and/or CASP10), it orchestrates apoptosis by catalyzing the cleavage of numerous proteins. In the early stages of apoptosis, it proteolytically cleaves poly(ADP-ribose) polymerase PARP1, targeting the '216-Asp-|-Gly-217' bond. CASP3 also activates sterol regulatory element-binding proteins (SREBPs), cleaves and activates caspase-6, -7, and -9, participates in the cleavage of huntingtin, and induces cell adhesion in sympathetic neurons through RET cleavage. Additionally, CASP3 cleaves and inhibits serine/threonine-protein kinase AKT1 in response to oxidative stress. It acts as an inhibitor of type I interferon production during virus-induced apoptosis by cleaving antiviral proteins CGAS, IRF3, and MAVS, thus preventing cytokine overproduction. CASP3 is also involved in pyroptosis, mediating the cleavage and activation of gasdermin-E (GSDME). Furthermore, it cleaves XRCC4 and phospholipid scramblase proteins XKR4, XKR8, and XKR9, promoting phosphatidylserine exposure on the apoptotic cell surface.

Measured by its ability to cleave the fluorogenic peptide substrate Ac-DEVD-AFC. The specific activity is ≥26 U/mg, as measured under the described conditions.
1U: The amount of enzyme required to consume l μmol of substrate per minute at 25°C.

Materials
Assay buffer: 25 mM HEPES, 0.1% (w/v) CHAPS, 10 mM dithiothreitol (DTT) , pH 7.5
Test protein: Caspase-3/CASP3 Protein, Human (HY-P701341)
Substrate: Ac-DEVD-AFC (HY-P1005), stored as a 10 mM solution diluted in DMSO.
Standard：7-amino, 4-(trifluoromethyl) coumarin (HY-D0981)

Procedure
1. Dilute 7-amino,4-(trifluoromethyl) coumarin in assay buffer to concentrations of 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μmol/L. Add 100 μL of each dilution to a black microplate well. Measure fluorescence at excitation and emission wavelengths of 400 nm and 505 nm, respectively, in kinetic mode for 5 minutes. Plot the measured RFU values on the y-axis and the standard concentrations on the x-axis to generate a standard curve and obtain the curve equation.
2. Dilute Human Caspase 3 to 0.4 μg/mL in assay buffer.
3. Dilute the substrate to 100 μM in assay buffer.
4. Experimental group: Add 50 μL of 0.4 μg/mL Human Caspase 3 to the plate, then add 50 μL of 100 μM substrate to initiate the reaction.
Control group: Add 50 μL of assay buffer and 50 μL of 100 μM substrate.
5. Read in kinetic mode for 5 minutes at excitation and emission wavelengths of 400 nm and 505 nm, respectively.
6. Calculate Specific Activity:

Human

E. coli

Tag Free

P42574 (S29-D175&S176-H277)

836

Full Length of Mature Protein

Approximately 12.6 kDa&16.6 kDa

≥ 90%, as determined by reducing SDS-PAGE.

Solution

Supplied as a 0.22 μm filtered solution of 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 20% glycerol, 1 mM DTT.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

Please use rapid thawing with running water to thaw the protein.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.