Active Caspase-3 antibody

Overview


	Antigen:	Active Caspase-3
	Antibody Type:	Monoclonal
	Application:	Immunoprecipitation (IP)
	Host:	Mouse, Rat
	Quantity:	100 µg (1 mg/mL)
	Price:	241,00 € (Plus shipping costs and VAT)
	Order number:	ABIN131778
	Availability:	7 to 10 days until shipment

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Additional Information: Active Caspase-3 antibody

Antigen	Active Caspase-3
Immunogen	This antibody was purified from hybridoma (clone AMI-3-1-11) supernatant using protein A agarose. This hybridoma was established by fusion of mouse myeloma cell Sp2/0 with Balb/c mouse splenocyte immunized with full length human recombinant Caspase-3.
Antibody Type	Monoclonal
Isotype	IgG2a »Matching secondary antibodies
Description	Caspase-3 (also known as CPP32, Yama, 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-caspase3 is processed at aspartate residues by self-proteolysis and/or cleavage by upstream caspases, such as caspase-6, 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 caspase3 proteolytically cleaves 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 those linked to mitochondrial release of cytochrome c.
Clone	AMI-3-1-11
Host	Mouse, Rat
Specificity	This antibody immunoprecipitates Caspase-3.

Application Details: Active Caspase-3 antibody

Application	Immunoprecipitation (IP)
Application Notes	Immunoprecipitation 1) Wash the cells 3 times with PBS and suspend with 10 volume 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. 3) Add primary antibody as suggested in the Applications into 400 µL of the supernatant. Mix well and incubate with gentle agitation for 30-120 minutes at 4 o C. Add 20 µL of 50% protein A agarose beads resuspended in the cold Lysis buffer. Mix well and incubate with gentle agitation for 60 minutes at 4 o C. 4) Wash the beads 3-5 times with the cold Lysis buffer (centrifuge the tube at 2,500 x g for 10 seconds). 5) Resuspend the beads in 20 µL of Laemmli’s sample buffer, boil for 3-5 minutes, and centrifuge for 5 minutes. Use 10 µL/lane for the SDS-PAGE analysis. 6) 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. 7) 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. 8) Incubate the membrane with the 10 µg / mL anti-caspase-3 antibody (MBL, code no.K0197-3) diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 9) Wash the membrane with PBS (5 minutes x 6 times). 10) Incubate the membrane with the 1:10,000 HRP-conjugated anti-mouse IgG (MBL code no. 330) diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 11) Wash the membrane with PBS (5 minutes x 6 times). 12) 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. 13) Expose to X-ray film in a dark room for 3 minutes. Develop the film as usual. The conditions for exposure and development may vary. Positive control for immunoprecipitation, Jurkat 050622-1 Caspase-3 Heavy chain Light chain 1 2 20 30 45 66 kDa Im m unoprecipita tion of C aspase-3 from Jurkatcells w ith n o rm a l m o u s e IgG 2a (1) or K0197-3 (2). After im m unoprecipita te d w ith the antibody, im m unocom plex w as resolved on SD S-PA G E and immunoblo tte d w ith K0197-3. Caspase-3 Heavy chain Light chain 1 2 20 30 45 66 kDa Caspase-3 Caspase-3 Heavy chain Heavy chain Light chain Light chain 1 2 20 30 45 66 kDa 1 2 20 30 45 66 20 30 45 66 kDa Im m unoprecipita tion of C aspase-3 from Jurkatcells w ith n o rm a l m o u s e IgG 2a (1) or K0197-3 (2). After im m unoprecipita te d w ith the antibody, im m unocom plex w as resolved on SD S-PA G E and immunoblo tte d w ith Western blotting: Not recommended. Immunoprecipitation: 10 µg / 400 µL of cell extract from 5x10 6 cells. Immunohistochemistry: Not recommended. Immunocytochemistry: Not tested. Flow Cytometry: Not tested.
Concentration	1 mg/mL
Buffer	100 µg IgG in 100 µL volume of PBS containing 50% glycerol, pH 7.2. Contains no preservatives.
Restrictions	For Research Use Only

Publications: Active Caspase-3 antibody

Publications

Yang, Fang, Wang et al.:
"Effect of NF-kappaB, survivin, Bcl-2 and Caspase3 on apoptosis of gastric cancer cells induced by tumor necrosis factor related apoptosis inducing ligand."
In: World journal of gastroenterology : WJG , Vol. 10, Issue 1, pp. 22-5, 2003/01 (PubMed)

Images: Active Caspase-3 antibody

Immunoprecipitation of Caspase-3 from Jurkat cells with normal mouse IgG2a (1) or ABIN131778 (2). After immunoprecipitated with the antibody, immunocomplex w as resolved on SDS -PAGE and immunoblotted with ABIN131778.

Image for Active Caspase-3 antibody (1)

External Information: Active Caspase-3 antibody

Google Scholar	Find more references for clone AMI-3-1-11 on Google Scholar™
EMBL Harvester	Search human, mouse or rat genome for antigen Active Caspase-3 (Bioinformatic Harvester (EMBL))