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Recombinant Human Caspase-3 Protein Summary
Details of Functionality
Measured by its ability to cleave the fluorogenic peptide substrate Ac-DEVD-AFC. The specific activity is >3,000 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived human Caspase-3 protein Ser29-Asp175 (subunit 1) & Ala183-His277 (Asp190Glu) (subunit 2)
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.
Applications/Dilutions
Dilutions
Enzyme Activity
Theoretical MW
17 kDa (subunit 1), 11 kDa (subunit 2). Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
18 kDa and 10 kDa, reducing conditions
Publications
Read Publications using 707-C3 in the following applications:
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
6 months from date of receipt, -70 °C as supplied.
3 months, -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in HEPES, NaCl, DTT and Sucrose with BSA as a carrier protein.
Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Assay Procedure
Assay Buffer: 25 mM HEPES, 0.1% (w/v) CHAPS, 10 mM dithiothreitol (DTT), pH 7.5
Recombinant Human Caspase-3 (rhCaspase-3) (Catalog # 707-C3)
Substrate: Ac-Asp-Glu-Val-Asp-AFC (MP Biomedicals, Catalog # AFC138), 10 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute rhCaspase-3 to 0.4 ng/µL in Assay Buffer.
Dilute Substrate to 100 µM in Assay Buffer.
Load 50 µL of 0.4 ng/µL rhCaspase-3 into a plate, and start the reaction by adding 50 µL of 100 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 100 µM Substrate.
Read at excitation and emission wavelengths of 400 nm and 505 nm (top read), respectively, in kinetic mode for 5 minutes.
Calculate specific activity:
Specific Activity (pmol/min/µg) =
Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)
amount of enzyme (µg)
*Adjusted for Substrate Blank
**Derived using calibration standard 7-amino, 4-(trifluoromethyl)coumarin (Calbiochem, Catalog #164580).
Per Well:
rhCaspase-3: 0.02 µg
Substrate: 50 µM
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Caspase-3 Protein
Apopain
apoptosis-related cysteine protease
CASP3
CASP-3
caspase 3, apoptosis-related cysteine peptidase
Caspase3
Caspase-3
CPP32
CPP-32
CPP32B
CPP32SREBP cleavage activity 1
Cysteine protease CPP32
EC 3.4.22
EC 3.4.22.56
LICE-1
PARP cleavage protease
procaspase3
Protein Yama
SCA-1
YAMA
Background
Caspase-3 (Cysteine-aspartic acid protease 3/Casp3; also Yama, apopain and CPP32) is a 29 kDa member of the peptidase C14A family of enzymes (1, 2, 3). It is widely expressed and is an integral component of the apoptotic cascade. Caspase-3 is considered to be the major executioner caspase; that is, the primary downstream mediator of apoptotic-associated proteolysis (2, 3, 4). Active Caspase-3 is known to utilize a Cys residue to cleave multiple substrates, including PARP, proIL‑16, PKC-gamma & -δ, procaspases 6, 7 and 9, and beta ‑catenin (1). Human procaspase-3 is a 32 kDa, 277 amino acid (aa) protein (5, 6, 7). Normally, it is an inactive, cytosolic homodimer, but following an upstream signal that activates processing proteases, procaspase-3 undergoes proteolytic cleavage (1, 2, 8, 9). This generates an N-terminal 175 aa p20/20 kDa subunit plus a 102 aa C-terminal p12/12 kDa subunit, followed by further processing of the p20 subunit at Asp28 to generate a final p17 subunit (aa 29‑175) (9). The p17 and p12 subunits noncovalently heterodimerize, and subsequently associate with another p17/p12 heterodimer to form an active antiparallel homodimer. The p17 subunit contains the enzyme active site (aa 161‑165), with an embedded catalytic Cys which is normally nitrosylated and inactive. Full activation requires both proteolytic processing and Cys163 denitrosylation (10). Multiple proteases can use Caspase-3 as a substrate including Caspase-6, -8, and -10, granzyme B, and Caspase-3 itself (9, 11, 12, 13).
Chowdhury, I. et al. (2008) Comp. Biochem. Physiol. B 151:10.
The Ins and Outs of Survivin By Rachel M.A. Linger, Ph.D.What is survivin?Survivin is a small (16.5 kDa) protein normally found in human fetal tissue. In contrast, survivin is typically undetectable in most normal adult tissues. Expression of ... Read full blog post.
Cleaved Caspase-3: A Marker of Programmed Cell Death What are Caspases?Caspases, or cysteine-dependent aspartate specific proteases, are a family of enzymes crucial for initiating and executing apoptosis within a cell, an important biological event especially during organ development (1). Environme... Read full blog post.
Caspase-3, The Executioner of Apoptosis The Role of Caspase-3 in ApoptosisCaspase-3 enzyme is a member of the family of endoproteases which regulate inflammation and apoptosis signaling networks. Caspase-3 is known as an executioner caspase in apoptosis because of its role in coordinat... Read full blog post.
Caspase 7 - A key effector of the apoptotic pathway Caspase-7 is an effector caspase with important roles in mediating cell death signaling. As an effector caspase, caspase-7 is cleaved and activated by initiator caspases such as caspase-1 (1). Like other caspase family proteins, caspase-7 contains a... Read full blog post.
D4-GDI (GDP dissociation inhibitor, RhoGD12) The D4-GDI protein is a negative regulator of the Ras-related Rho family of small molecule "molecular switch" GTPases. The Rho GTPases modify cell structure and architecture via rapid changes to the actin cytoskeleton and cell membrane. M... Read full blog post.
LC3B - a novel marker for autophagosome Autophagy, also known as macroautophagy, supplies alternative fuel for cells that are under environmental stress conditions (including starvation, growth factor deprivation, and hypoxia). This highly regulated and catabolic cell process recycles a... Read full blog post.
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