Recombinant Human Caspase-3 Protein, CF

R&D Systems | Catalog # 707-C3/CF

R&D Systems

Key Product Details

  • R&D Systems E. coli-derived Recombinant Human Caspase-3 Protein (707-C3/CF)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

P42574

Applications

Enzyme Activity
View all Caspase-3 Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human Caspase-3 protein
Ser29-Asp175 (subunit 1) & Ala183-His277 (Asp190Glu) (subunit 2)

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Ser29 (subunit 1) & Ala183 (subunit 2)

Predicted Molecular Mass

17 kDa (subunit 1), 11 kDa (subunit 2)

SDS-PAGE

16 kDa and 8 kDa, reducing conditions

Activity

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.

Formulation, Preparation, and Storage

707-C3/CF
Formulation Supplied as a 0.2 μm filtered solution in HEPES, NaCl, DTT and Sucrose.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage 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.

Background: Caspase-3

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 & -delta, 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). 

References

  1. Chowdhury, I. et al. (2008) Comp. Biochem. Physiol. B 151:10.
  2. Boatright, K.M. & G.S. Salvesen (2003) Curr. Opin. Cell Biol. 15:725.
  3. Launay, S. et al. (2005) Oncogene 24:5137.
  4. Walsh, J.G. et al. (2008) Proc. Natl. Scad. Sci. USA 105:12815.
  5. Nicholson, D.W. et al. (1995) Nature 376:37.
  6. Tewari, M. et al. (1995) Cell 81:801.
  7. Fernandes-Alnemri, T. et al. (1994) J. Biol. Chem. 269:30761.
  8. Milisav, I. et al. (2009) Apoptosis 14:1070.
  9. Han, Z. et al. (1997) J. Biol. Chem. 272:13432.
  10. Rossig, L. et al. (1999) J. Biol. Chem. 274:6823.
  11. Rank, K.B. et al. (2001) Protein Expr. Purif. 22:258.
  12. Atkinson, E.A. et al. (1998) J. Biol. Chem. 273:21261.
  13. Cohen, G.M. (1997) Biochem. J. 326:1.

Alternate Names

Apopain, CASP3, Caspase3, CPP32, LICE-1, YAMA

Entrez Gene IDs

836 (Human); 12367 (Mouse)

Gene Symbol

CASP3

UniProt

P42574 (Rabbit)

Additional Caspase-3 Products

Product Documents for Recombinant Human Caspase-3 Protein, CF

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human Caspase-3 Protein, CF

For research use only

Related Research Areas

Cysteine Proteases and Regulators

Citations for Recombinant Human Caspase-3 Protein, CF

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Protocols

View specific protocols for Recombinant Human Caspase-3 Protein, CF (707-C3/CF):

Materials
  • 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/CF)
  • 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
  1. Dilute rhCaspase 3 to 0.4 ng/µL in Assay Buffer.
  2. Dilute Substrate to 100 µM in Assay Buffer.
  3. 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.
  4. Read at excitation and emission wavelengths of 400 nm and 505 nm (top read), respectively, in kinetic mode for 5 minutes.
  5. 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

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