Recombinant Human Caspase-3 Protein

• Reactivity: Hu
• Applications: Enzyme Activity

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)
• Accession #: P42574
• N-terminal Sequence: Ser29 (subunit 1) & Ala183 (subunit 2)
• Protein/Peptide Type: Recombinant Enzymes
• Gene: CASP3
• Purity: >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

• 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

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 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
  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

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). 

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.

Publications for Caspase-3 (707-C3)(8)

Publications using 707-C3

• B Zhou, JY Zhang, XS Liu, HZ Chen, YL Ai, K Cheng, RY Sun, D Zhou, J Han, Q Wu Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis Cell Res., 2018;0(0):. 2018 [PMID: 30287942] (Bioassay, Human)
• F Wang, L Wang, J Wu, I Sokirniy, P Nguyen, T Bregnard, J Weinstock, M Mattern, I Bezsonova, WW Hancock, S Kumar Active site-targeted covalent irreversible inhibitors of USP7 impair the functions of Foxp3+ T-regulatory cells by promoting ubiquitination of Tip60 PLoS ONE, 2017;12(12):e0189744. 2017 [PMID: 29236775] (Bioassay, Human)
• MK Shim, HY Yoon, S Lee, MK Jo, J Park, JH Kim, SY Jeong, IC Kwon, K Kim Caspase-3/-7-Specific Metabolic Precursor for Bioorthogonal Tracking of Tumor Apoptosis Sci Rep, 2017;7(1):16635. 2017 [PMID: 29192289] (EnzAct, Human)
• Fluorogenic Substrates for In Situ Monitoring of Caspase-3 Activity in Live Cells PLoS ONE, 2016;11(5):e0153209. 2016 [PMID: 27168077] (Bioassay, Human)
• 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;287(41):33983-95. 2012 [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;118(17):4694-704. 2011 [PMID: 21876121] (Enzyme Assay, Human)
• Ghosh JG, Shenoy AK, Clark JI Interactions between important regulatory proteins and human alphaB crystallin. Biochemistry, 2007;46(21):6308-17. 2007 [PMID: 17487982] (ELISA-based Protein Pin Array, Human)
• Lane D, Cote M, Grondin R, Couture MC, Piche A Acquired resistance to TRAIL-induced apoptosis in human ovarian cancer cells is conferred by increased turnover of mature caspase-3. Mol. Cancer Ther., 2006;5(3):509-21. 2006 [PMID: 16546965] (EnzAct, Human)

Bioinformatics

• Gene Symbol: CASP3
• Uniprot:
  • P42574()