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.
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.
Preparation and Storage
Reconstitute at 0.2 mg/mL in sterile PBS.
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C
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.
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.
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