Recombinant Human Pro-Caspase-3 Protein, CF

R&D Systems | Catalog # 731-C3

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human Pro-Caspase-3 Protein (731-C3)
  • 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

AAA65015

Structure / Form

Pro form

Applications

Enzyme Activity
View all Caspase-3 Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human Caspase-3 protein
Met1-His277, with an N-terminal Met and 6-His tag

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by silver stain.

N-terminal Sequence Analysis

Not tested: Met predicted.

Predicted Molecular Mass

33 kDa

SDS-PAGE

35 kDa, reducing conditions

Activity

The activity of the activated Caspase-3 was measured by its ability to cleave DEVD-AFC.
The specific activity is >500 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

731-C3
Formulation Lyophilized from a 0.2 μm filtered solution in Tris, NaCl, Sucrose and DTT.
Reconstitution Reconstitute at 50 µg/mL in 20 mM Tris, 300 mM NaCl, 5 mM Dithiothreitol, 5% Sucrose and 0.05% CHAPS, pH 8.0.
Shipping The product is shipped at ambient temperature. 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, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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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

AAA65015

Additional Caspase-3 Products

Product Documents for Recombinant Human Pro-Caspase-3 Protein, CF

Certificate of Analysis

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

For research use only

Related Research Areas

Cysteine Proteases and Regulators

Citations for Recombinant Human Pro-Caspase-3 Protein, CF

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Protocols

View specific protocols for Recombinant Human Pro-Caspase-3 Protein, CF (731-C3):

Materials
  • Assay Buffer: 20 mM Tris, 0.3 M NaCl, 0.05% (w/v) CHAPS, 5 mM DTT, 5% (w/v) Sucrose, pH 8.0
  • Recombinant Human Pro-Caspase-3 (rhPro-Caspase-3) (Catalog # 731-C3)
  • Recombinant Human Caspase‑8 (rhCaspase-8) (Catalog # 705-C8)
  • 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. Activate rhPro-Caspase-3 at 10 μg/mL with 10 µg/mL rhCaspase-8 at a final concentration, respectively in Assay Buffer.
  2. Incubate reaction at 37 °C for 90 minutes.
  3. Dilute Substrate to 100 µM in Assay Buffer.
  4. Dilute Activated rhPro-Caspase-3 to 0.4 µg/mL.
  5. In a plate load 50 µL of 0.4 µg/mL rhPro-Caspase-3 and include a Substrate Blank containing 50 µL Assay Buffer.
  6. Start the reaction by adding 50 µL of 100 µM Substrate to wells.
  7. Read at excitation and emission wavelengths of 400 nm and 505 nm (top read), respectively in kinetic mode for 5 minutes.
  8. 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 (Biomedicals, Catalog # 164580).

Per Well:

  • rhPro-Caspase-3: 0.02 µg
  • Substrate: 50 µM

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