Caspase 3 antibody

Catalog No. ABIN5541795

The Rat Monoclonal anti-Caspase 3 antibody has been validated for WB. It is suitable to detect Caspase 3 in samples from Human.

Quick Overview for Caspase 3 antibody (ABIN5541795)

Target: Caspase 3 (CASP3)

Reactivity: Human

Host: Rat

Clonality: Monoclonal

Conjugate: This Caspase 3 antibody is un-conjugated

Application: Western Blotting (WB)

Clone: 1F3

Product Details anti-Caspase 3 Antibody

Specificity: This antibody reacts with Pro-Caspase-3 (32 kDa) and active Caspase-3 (17 kDa).

Purification: Protein G agarose

Immunogen: Full length human recombinant Caspase-3

Isotype: IgG2a

Application Details

Application Notes: Western blot: 1 μg / ml for chemiluminescence detection system. For details see protocol below. Not recommended for Immunoprecipitatoin or Immunohistochemistry.

Protocol: SDS-PAGE & Western Blotting 1) Wash the cells 3 times with PBS and suspend with 10 volumes of cold Lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1 % NP-40, 2 mM EDTA, 10 % glycerol) containing appropriate protease inhibitors. Incubate it at 4 o C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 o C and transfer the supernatant to another tube. Measure the protein concentration of the supernatant and add the cold Lysis buffer to make 8 mg/mL solution. 3) Mix the sample with equal volume of Laemmli's sample buffer. 4) Boil the samples for 2 minutes and centrifuge. Load 10 μ L of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20 % MeOH). See the manufacture's manual for precise transfer procedure. 6) To reduce nonspecific binding, soak the membrane in 10 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4 o C. 7) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 1 % skimmed milk as suggested in the APPLICATIONS for 1 hour at room temperature. (The optimal antibody concentration will depend on the experimental conditions.) 8) Wash the membrane with PBS (5 minutes x 6 times). 9) Incubate the membrane with the 1:10,000 HRP-conjugated anti-rat IgG diluted with 1 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 10) Wash the membrane with PBS (5 minutes x 6 times). 11) Wipe excess buffer from the membrane, then incubate it with appropriate chemiluminescence reagents for 1 minute. Remove extra reagent from the membrane by dabbing with a paper towel, and seal it in plastic wrap. 12) Expose to X-ray film in a dark room for 5 minutes. Develop the film as usual. The conditions for exposure and development may vary. Positive controls for Western blotting Jurkat and Apoptotic Jurkat

Restrictions: For Research Use only

Handling

Format: Liquid

Buffer: PBS containing 50 % glycerol, pH 7.2. Contains no preservatives.preservatives.

Preservative: Without preservative

Storage: -20 °C

Storage Comment: Upon receipt, store undiluted (in aliquots) at -20°C. Avoid repeated freezing and thawing. Shelf life: One year from despatch.

Target Details for Caspase 3

Target: Caspase 3 (CASP3)

Alternative Name: caspase-3

Background: Caspase-3 (also known as CPP32, Yama, and apopain) is a key member of the caspase family of cysteine proteases. Caspase-3 exists in cells as an inactive 32 kDa proenzyme. During apoptosis pro-caspase-3 is processed at aspartate residues by self-proteolysis and/or cleav age by upstream caspases, such as caspase6, 8, or 9. The processed form of caspase-3 consists of large (17 kDa) and small (12 kDa) subunits which associate to form the active tetrameric enzyme tetramer (a pair of heterodimers). The active caspase-3 proteolytically cl eaves and activates other caspases, as well as relevant targets in the cells (e.g., PARP, SREBPs, and DFF). Activation of pro-caspase-3 stands at a point of convergence for the two major types of apoptosis signaling pathways-those linked to cell surface death receptors and th ose linked to mitochondrial release of cytochrome c.

UniProt: P42574

Pathways: Apoptosis, Caspase Cascade in Apoptosis, Sensory Perception of Sound, ER-Nucleus Signaling, Positive Regulation of Endopeptidase Activity, Activated T Cell Proliferation