Caspase-3 Antibody [Unconjugated]

Western blot shows lysates of Jurkat human acute T cell leukemia cell line and DA3 mouse myeloma cell line untreated (-) or treated (+) with 1 µg/mL Staurosporine (STS) for 12 hours. PVDF Membrane was probed with 0.5 µg/mL of Goat Anti-Human Caspase‑3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-605-NA) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). Specific bands were detected for Caspase‑3 precursor at approximately 34 kDa (as indicated) and Caspase‑3 (cleaved) at approximately 18 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 2.

Jurkat human acute T cell leukemia cell line was treated with indicated concentrations of Staurosporine for 0 or 4 hours. Caspase‑3 was immunoprecipitated from lysates of 1‑2 x 106 cells following incubation with 1 or 4 µg Goat Anti-Human Caspase‑3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-605-NA) overnight at 4 ºC. Caspase‑3-antibody complexes were absorbed using Protein G expressing Staph cells (Sigma). Immunoprecipitated Caspase‑3 was detected by Western blot using 0.5 µg/mL Goat Anti-Human Caspase‑3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF‑605‑NA). View our recommended buffer recipes for immunoprecipitation.

Simple Western lane view shows lysates of HeLa human cervical epithelial carcinoma cell line, HepG2 human hepatocellular carcinoma cell line, and Jurkat human acute T cell leukemia cell line, loaded at 0.2 mg/mL. A specific band was detected for Caspase‑3 at approximately 40 kDa (as indicated) using 5 µg/mL of Goat Anti-Human/Mouse Caspase‑3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-605-NA) 1:50 dilution followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and Caspase-3 knockout HeLa cell line (KO). PVDF membrane was probed with 0.2 µg/mL of Goat Anti-Human/Mouse Caspase‑3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-605-NA) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for Caspase‑3 at approximately 32 kDa (as indicated) in the parental HeLa cell line, but is not detectable in knockout HeLa cell line. GAPDH (Catalog # AF5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

• Reactivity: Hu, Mu
• Applications: WB, Simple Western, IP, KO
• Clonality: Polyclonal
• Host: Goat
• Conjugate: Unconjugated
• Concentration: LYOPH

Caspase-3 Antibody [Unconjugated] Summary

• Immunogen: E. coli-derived recombinant human Caspase-3
  Met1-His277
  Accession # AAA65015
• Specificity: Detects human Caspase-3.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Goat
• Gene: CASP3

Applications/Dilutions

• Dilutions:
  • Western Blot 0.5 ug/mL
  • Simple Western 5 ug/mL
  • Immunoprecipitation 1 ug/10^6 cells
  • Knockout Validated
• Application Notes: In Simple Western only 10-15 uL of the recommended dilution is used per data point.

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile PBS.

Notes

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

Alternate Names for Caspase-3 Antibody [Unconjugated]

• 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.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for Caspase-3 Antibody (AF-605-NA)(6)

Publications using AF-605-NA

• M Biernacki, W ?uczaj, I Jarocka-Ka, E Ambro?ewic, M Toczek, E Skrzydlews The Effect of Long-Term Administration of Fatty Acid Amide Hydrolase Inhibitor URB597 on Oxidative Metabolism in the Heart of Rats with Primary and Secondary Hypertension Molecules, 2018;23(9):. 2018 [PMID: 30223427] (Western Blot, Rat)
• S Einsele-Sc, S Malmsheime, K Bertram, D Stehle, J Johänning, M Manz, PT Daniel, BF Gillissen, K Schulze-Os, F Essmann Bok is a genuine multi-BH-domain protein that triggers apoptosis in the absence of Bax and Bak and augments drug response J Cell Sci, 2016;0(0):. 2016 [PMID: 27076518] (Western Blot, Human)
• Deng J, Han Y, Yan C, Tian X, Tao J, Kang J, Li S Overexpressing cellular repressor of E1A-stimulated genes protects mesenchymal stem cells against hypoxia- and serum deprivation-induced apoptosis by activation of PI3K/Akt. Apoptosis, 2010;15(4):463-73. 2010 [PMID: 19997978] (Western Blot, Rat)
• Tian E, Landowski TH, Stephens OW, Yaccoby S, Barlogie B, Shaughnessy JD Ellipticine derivative NSC 338258 represents a potential new antineoplastic agent for the treatment of multiple myeloma. Mol. Cancer Ther., 2008;7(3):500-9. 2008 [PMID: 18319333] (Western Blot, Human)
• Engesaeter BØ, Bonsted A, Lillehammer T, Engebraaten O, Berg K, Maelandsmo GM Photochemically mediated delivery of AdhCMV-TRAIL augments the TRAIL-induced apoptosis in colorectal cancer cell lines. Cancer Biol. Ther., 2006;5(11):1511-20. 2006 [PMID: 17012835] (Western Blot, Human)
• Chae JJ, Wood G, Masters SL, Richard K, Park G, Smith BJ, Kastner DL The B30.2 domain of pyrin, the familial Mediterranean fever protein, interacts directly with caspase-1 to modulate IL-1beta production. Proc. Natl. Acad. Sci. U.S.A., 2006;103(26):9982-7. 2006 [PMID: 16785446] (Western Blot, Human)

Bioinformatics

• Gene Symbol: CASP3
• Uniprot:
  • AAA65015()