Caspase-3 Antibody

R&D Systems | Catalog # AF-605-NA

R&D Systems
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Key Product Details

Validated by

Knockout/Knockdown, Biological Validation

Species Reactivity

Validated:

Human, Mouse

Cited:

Human, Mouse, Rat, Insect - Drosophila melanogaster, Transgenic Mouse

Applications

Validated:

Knockout Validated, Western Blot, Simple Western, Immunoprecipitation

Cited:

Immunohistochemistry, Western Blot, Immunocytochemistry, Proximity Extension Assay

Label

Unconjugated

Antibody Source

Polyclonal Goat IgG
View all Caspase-3 Primary Antibodies »

Product Specifications

Immunogen

E. coli-derived recombinant human Caspase-3
Met1-His277
Accession # AAA65015

Specificity

Detects human Caspase-3.

Clonality

Polyclonal

Host

Goat

Isotype

IgG

Scientific Data Images for Caspase-3 Antibody

Detection of Human and Mouse Caspase-3 antibody by Western Blot.

Detection of Human and Mouse Caspase‑3 by Western Blot.

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.
Immunoprecipitation of Human Caspase‑3.

Immunoprecipitation of Human Caspase‑3.

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 106cells 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.
Detection of Human Caspase-3 antibody by Simple WesternTM.

Detection of Human Caspase‑3 by Simple WesternTM.

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 Human Caspase-3 Antibody Specificity by Using Knockout Cell Line.

Western Blot Shows Human Caspase‑3 Specificity by Using Knockout Cell Line.

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.
Detection of Caspase-3 by Western Blot

Detection of Caspase-3 by Western Blot

Effect of STZ injection and MitoTEMPOL on carbonyl and caspase protein levels. (A) Protein carbonylation was increased in the diabetic group compared with the other groups. (B) Representative protein immunoblot of caspase 3, cleaved caspase 3, caspase 9, and cleaved caspase 9 protein. (C) Cleaved caspase 3 protein level was significantly reduced in the diabetic + TEMPOL group compared to the diabetic group only. (D) Cleaved caspase 9 protein level was significantly reduced in the diabetic + TEMPOL group compared to the diabetic group only. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35888150), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Caspase-3 Antibody

Application
Recommended Usage

Immunoprecipitation

1 µg/106 cells
Sample: Jurkat human acute T cell leukemia cell line treated with Staurosporine, see our available Western blot detection antibodies

Knockout Validated

Caspase‑3 is specifically detected in HeLa human cervical epithelial carcinoma parental cell line but is not detectable in Caspase‑3 knockout HeLa cell line.

Simple Western

5 µg/mL
Sample: HeLa human cervical epithelial carcinoma cell line, HepG2 human hepatocellular carcinoma cell line, and Jurkat human acute T cell leukemia cell line

Western Blot

0.5 µg/mL
Sample: Jurkat human acute T cell leukemia cell line and DA3 mouse myeloma cell line treated with Staurosporine

Reviewed Applications

Read 1 review rated 4 using AF-605-NA in the following applications:

Formulation, Preparation, and Storage

Purification

Antigen Affinity-purified

Reconstitution

Reconstitute at 0.2 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & 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.

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 Caspase-3 Antibody

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Caspase-3 Antibody

For research use only

Related Research Areas

Cysteine Proteases and Regulators

Citations for Caspase-3 Antibody

Customer Reviews for Caspase-3 Antibody (1)

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  • Human/Mouse Caspase-3 Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: A549 human lung carcinoma cell line
    Species: Human
    Verified Customer | Posted 04/08/2022
    Caspase-3 Antibody AF-605-NA

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