Caspase-3/CASP3 Protein, Human (His)

Caspase-3/CASP3 protein is critical in the execution of apoptosis, cleaving PARP1 and activating SREBP, caspase-6, -7 and -9, initiating the cascade leading to apoptosis. In addition to apoptosis, it regulates cell adhesion, promotes inflammation, inhibits AKT1 in oxidative stress, and modulates antiviral responses by cleaving proteins such as CGAS, IRF3, and MAVS. Caspase-3/CASP3 Protein, Human (His) is the recombinant human-derived Caspase-3/CASP3 protein, expressed by E. coli , with C-His labeled tag.

Caspase-3, a pivotal player in the apoptosis execution pathway, holds a central role in diverse cellular processes. During the initiation of apoptosis, it orchestrates the proteolytic cleavage of poly(ADP-ribose) polymerase PARP1, exemplifying its regulatory influence over critical cellular components. This versatile protein extends its reach by cleaving and activating sterol regulatory element-binding proteins (SREBPs), caspase-6, -7, and -9, thus participating in a cascade of events leading to apoptosis. Beyond its classical apoptotic functions, Caspase-3 exhibits involvement in cell adhesion within sympathetic neurons through RET cleavage and contributes to the inflammatory milieu by cleaving IL-1 beta. In response to oxidative stress, it inhibits the serine/threonine-protein kinase AKT1. Additionally, Caspase-3 assumes a role in regulating antiviral responses by cleaving proteins such as CGAS, IRF3, and MAVS, curbing type I interferon production. Furthermore, it plays a role in pyroptosis by cleaving and activating gasdermin-E (GSDME), as well as modulating phosphatidylserine exposure on apoptotic cell surfaces through XRCC4 and phospholipid scramblase protein cleavage. This comprehensive range of actions underscores the intricate regulatory functions of Caspase-3 in cellular homeostasis and response to diverse stimuli.

Measured by its ability to cleave the fluorogenic peptide substrate Ac-DEVD-AFC (HY-P1005) and the specific activity is > 5000 pmoles/min/μg. (Activation description: The proenzyme needs to be activated by Caspase-8 for an activated form.)

Materials
Assay Buffer: 25 mM HEPES, 0.1% (w/v) CHAPS, 10 mM dithiothreitol (DTT) , pH 7.5.
Caspase-3/CASP3 Protein, Human (His) (HY-P72874)
Caspase-8, Human
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. Standard Curve Preparation：Dilute standard 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 in kinetic mode for 5 minutes at excitation and emission wavelengths of 400 nm and 505 nm, respectively. 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. Activate 10 μg/mL of Human Caspase-3 with 10 μg/mL of Human Caspase-8 in the assay buffer.
3. Incubate at 37°C for 90 minutes.
4. Dilute the substrate to 100 μM in the assay buffer.
5. Add 50 μL of 0.4 μg/mL Human Caspase-3 to the plate and include a substrate blank containing 50 μL of assay buffer. Initiate the reaction by adding 50 μL of 100 μM substrate to the wells.
6. Read in kinetic mode for 5 minutes at excitation and emission wavelengths of 400 nm and 505 nm, respectively.
7. Calculate the specific activity:

Human

E. coli

C-6*His

P42574 (S29-H277)

836

Full Length of Mature Protein

SGISLDNSYKMDYPEMGLCIIINNKNFHKSTGMTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFVCVLLSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQACRGTELDCGIETDSGVDDDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADKLEFMHILTRVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFYH

Approximately 29 kDa

• 
  Greater than 95% as determined by reducing SDS-PAGE.

Lyophilized powder

Lyophilized from sterile PBS, pH 7.4, 8% trehalose.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O.

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Shipping with dry ice.