Recombinant Human Pro-Caspase-3 Protein, CF Summary
Met1-His277, with an N-terminal Met and 6-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|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.
- 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
- Activate rhPro-Caspase-3 at 10 μg/mL with 10 µg/mL rhCaspase-8 at a final concentration, respectively in Assay Buffer.
- Incubate reaction at 37 °C for 90 minutes.
- Dilute Substrate to 100 µM in Assay Buffer.
- Dilute Activated rhPro-Caspase-3 to 0.4 µg/mL.
- In a plate load 50 µL of 0.4 µg/mL rhPro-Caspase-3 and include a Substrate Blank containing 50 µL Assay Buffer.
- Start the reaction by adding 50 µL of 100 µM Substrate to wells.
- 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 (Biomedicals, Catalog # 164580).
- rhPro-Caspase-3: 0.02 µg
- Substrate: 50 µM
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.
Boatright, K.M. & G.S. Salvesen (2003) Curr. Opin. Cell Biol. 15:725.
Launay, S. et al. (2005) Oncogene 24:5137.
Walsh, J.G. et al. (2008) Proc. Natl. Scad. Sci. USA 105:12815.
Nicholson, D.W. et al. (1995) Nature 376:37.
Tewari, M. et al. (1995) Cell 81:801.
Fernandes-Alnemri, T. et al. (1994) J. Biol. Chem. 269:30761.
Milisav, I. et al. (2009) Apoptosis 14:1070.
Han, Z. et al. (1997) J. Biol. Chem. 272:13432.
Rossig, L. et al. (1999) J. Biol. Chem. 274:6823.
Rank, K.B. et al. (2001) Protein Expr. Purif. 22:258.
Atkinson, E.A. et al. (1998) J. Biol. Chem. 273:21261.
- Cohen, G.M. (1997) Biochem. J. 326:1.
Citations for Recombinant Human Pro-Caspase-3 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
Citations: Showing 1 - 2
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PLF-1 (Proliferin-1) Modulates Smooth Muscle Cell Proliferation and Development of Experimental Intimal Hyperplasia
Authors: L Hu, Z Huang, H Ishii, H Wu, S Suzuki, A Inoue, W Kim, H Jiang, X Li, E Zhu, L Piao, G Zhao, Y Lei, K Okumura, GP Shi, T Murohara, M Kuzuya, XW Cheng
J Am Heart Assoc, 2019;8(24):e005886.
Sample Types: Recombinant Protein
Proteinase 3-dependent caspase-3 cleavage modulates neutrophil death and inflammation.
Authors: Loison, Fabien, Zhu, Haiyan, Karatepe, Kutay, Kasorn, Anongnar, Liu, Peng, Ye, Keqiang, Zhou, Jiaxi, Cao, Shannan, Gong, Haiyan, Jenne, Dieter E, Remold-O'Donnell, Eileen, Xu, Yuanfu, Luo, Hongbo R
J Clin Invest, 2014;124(10):4445-58.
Sample Types: Cell Lysates
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