Recombinant Human Active Caspase-2 Protein

Product Discontinued

Recombinant Human Active Caspase-2 Protein Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate Ac-VDVAD-AFC. The specific activity is >1000 pmol/min/µg, as measured under the described conditions.
• Source: E. coli-derived human Caspase-2 protein
  Gly170-Asp333 & Ala348-Tyr452
• Accession #: P42575
• N-terminal Sequence: Gly170 & Ala348
• Protein/Peptide Type: Recombinant Enzymes
• Gene: CASP2
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 12 kDa & 18 kDa.
  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: 10 kDa & 17 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• 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 under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 25 mM HEPES, 0.1% (w/v) CHAPS, 10 mM DTT, pH 7.5
  • Recombinant Human Active Caspase-2 (rhCaspase-2) (Catalog # 702-C2)
  • Substrate: Ac-Val-Asp-Val-Ala-Asp-AFC (MP Biomedicals, Catalog # AFC142), 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-2 to 1 ng/µL in Assay Buffer.
  2. Incubate at room temperature for 15 minutes.
  3. Dilute Substrate to 200 µM with Assay Buffer.
  4. Load into plate 50 µL of 1 ng/µL rhCaspase-2 and start the reaction by adding 50 µL of 200 µM Substrate.
  5. Read at excitation and emission wavelengths of 400 nm and 505 nm (top read), respectively in kinetic mode for 5 minutes.
  6. 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-2: 0.050 µg
  • Substrate: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Active Caspase-2 Protein

• caspase-2
• CASP2
• CASP-2
• caspase 2, apoptosis-related cysteine peptidase
• Caspase2
• Caspase-2
• ICH1
• ICH-1
• NEDD2
• NEDD-2
• PPP1R57

Background

Caspase-2 (Cysteine-aspartic acid protease 2/Casp2; also NEDD2 and ICH-1) is a 30‑32 kDa member of the peptidase C14A/IL‑1 beta ‑converting family of enzymes (1‑3). It is widely expressed and is an integral component of the apoptotic cascade. Based on the length of its prodomain, caspase-2 has been considered to be an initiator caspase. However, studies have shown that other caspases (such as Casp 3) activate procaspase 2, and Caspase-2 likely acts on key cellular molecules such as BID, Golgin 160 and DFF45/ICAD (2, 4, 5). Thus, Caspase-2 is perhaps more likely to be a specialized executioner caspase. Human procaspase-2 is a 48‑51 kDa, 452 amino acid (aa) protein (4‑7). It is known to exist as a disulfide-linked homodimer via covalent linkage at Cys436 (2, 5). But this dimeric state may not be sufficient for (auto)activation. Actual activation may occur following oligomerization within the context of activating platforms such as DISC (death-inducing signaling complex) or the PIDDosome (8‑10). Initially, procaspase-2 undergoes proteolytic cleavage to generate an N-terminal 333 aa p34/34 kDa subunit, and a 119 aa C-terminal p14/14 kDa subunit (5). The p34 and p14 subunits are further processed to generate the prodomain (aa 1‑169), plus the mature p18 (aa 170‑333) and p12 (aa 348‑452) subunits (4‑6). Notably, each p18:p12 noncovalent heterodimer demonstrates proteolytic activity around a catalytic site at aa 318‑322, and, due to an nuclear localization signal within the prodomain, may be found in either nucleus or cytoplasm (11, 12). There are multiple potential isoform variants. Individually, or in combination, there is an alternative start site at Met18, a substitution of two aa for aa 107‑452, a second substitution of 14 aa for aa 309‑322, and a third substitution of 22 aa for aa 323‑452 (6, 7, 13). The human and mouse procaspase 2 precursors are 90% aa identical, with the majority of differences lying in the prodomain.

1. Chowdhury, I. et al. (2008) Comp. Biochem. Physiol. B 151:10.
2. Krumschnabel, G. et al. (2009) Cell Death Differ.16:195.
3. Kitevska, T. et al. (2009) Apoptosis 14:829.
4. Paroni, G. et al. (2001) J. Biol. Chem. 276:21907.
5. Li, H. et al. (1997) J. Biol. Chem. 272:21010.
6. SwissProt. Accession # P42575.
7. Wang, L. et al. (1994) Cell 78:739.
8. Chang, D.W. et al. (2003) J. Biol. Chem. 278:16466.
9. Olsson, M. et al. (2009) Oncogene 28:1949.
10. Tinel, A. & J. Tschopp (2004) Science 304:843.
11. Schweizer, A. et al. (2003) J. Biol. Chem. 278:42441.
12. Colussi, P.A. et al. (1998) J. Biol. Chem. 273:24535.
13. Droin, N. et al. (2000) Cancer Res. 60:7039.
 

Publications for Caspase-2 (702-C2)(3)

Publications using 702-C2

• Yun N, Lee Y, Kim C, Shibayama H, Tanimura A, Hamanaka Y, Kanakura Y, Park I, Jo A, Shin J, Ju C, Kim W, Oh Y Anamorsin, a novel caspase-3 substrate in neurodegeneration. J Biol Chem, 2014-06-27;289(32):22183-95. 2014-06-27 [PMID: 24973211] (Enzyme Assay, Mouse)
• Wejda M, Impens F, Takahashi N, Van Damme P, Gevaert K, Vandenabeele P Degradomics reveals that cleavage specificity profiles of caspase-2 and effector caspases are alike. J Biol Chem, 2012-07-23;287(41):33983-95. 2012-07-23 [PMID: 22825847] (Enzyme Assay, Human)
• Guery L, Benikhlef N, Gautier T, Paul C, Jego G, Dufour E, Jacquel A, Cally R, Manoury B, Vanden Berghe T, Vandenabeele P, Droin N, Solary E Fine-tuning nucleophosmin in macrophage differentiation and activation. Blood, 2011-08-29;118(17):4694-704. 2011-08-29 [PMID: 21876121] (Enzyme Assay, Human)

Bioinformatics

• Gene Symbol: CASP2
• Uniprot:
  • P42575()