Caspase 9 Antikörper (N-Term)

Produktnummer ABIN5541756

Der Maus Monoklonal Anti-Caspase 9-Antikörper wurde für WB validiert. Er ist geeignet, Caspase 9 in Proben von Human, Maus und Ratte zu detektieren.

Kurzübersicht für Caspase 9 Antikörper (N-Term) (ABIN5541756)

Target: Caspase 9 (CASP9) (Caspase 9, Apoptosis-Related Cysteine Peptidase (CASP9))

Reaktivität: Human, Maus, Ratte

Wirt: Maus

Klonalität: Monoklonal

Konjugat: Dieser Caspase 9 Antikörper ist unkonjugiert

Applikation: Western Blotting (WB)

Klon: 5B4

Produktdetails anti-Caspase 9 Antikörper

Bindungsspezifität: AA 1-250, N-Term

Spezifität: This antibody reacts with 45 kDa of pro-caspase-9 and cleaved 35 or 37 kDa products.

Aufreinigung: Protein A agarose

Immunogen: Recombinant protein corresponding to N-terminal amino acids (1-250 aa) of human caspase-9

Isotyp: IgG1

Anwendungsinformationen

Applikationshinweise: Western blot: 1 μg/mL for chemiluminescence detection system. For details see protocol bleow. Not recommended for Immunoprecipitation.

Protokoll: SDS-PAGE & Western Blotting 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. 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 3 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 5 % 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 5 % skimmed milk as suggest in the APPLICATIONS for 1 hour at room temperature. (The concentration of antibody will depend on condition.) 8) Wash the membrane with PBS-T [0.05 % Tween-20 in PBS] (10 minutes x 3 times). 9) Incubate the membrane with the 1:5,000 HRP-conjugated anti-mouse IgG diluted with 5 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 10) Wash the membrane with PBS-T (10 minutes x 3 times). 11) Wipe excess buffer on the membrane, then incubate it with appropriate chemiluminescen ce reagent for 1 minute. 12) Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap. 13) Expose to an X-ray film in a dark room for 2 minutes. 14) Develop the film as usual. The condition for exposure and development may vary. (Positive controls for Western blotting Jurkat, Raji, U937, NIH/3T3, Ba/F3, PC12) Detection of Cleaved Caspase-9 1) Prepare a 1 mM staurosporine stock solution by dissolving staurosporine in DMSO. 2) Collect 1 x 10 7 of semi-confluently grown Jurkat cells by centrifugation, remove the medium and resuspend with 10 mL of growing medium containing 1 μ M of staurosporine. 3) Incubate the cell suspension for 4 hours at 37 °C. Harvest the cells by centrifugation. 4) Rinse the cells twice with PBS and resuspend in 1 mL of Laemmli's sample buffer. 5) Lyse the cells by brief sonication (up to 10 seconds) and boil for 5 minutes. Centrifuge it at 12000 x g for one minute. 6) Use 5~20 μ L/lane of the sample for the SDS-PAGE and Western blotting analysis. (See SDS-PAGE & Western blotting. Induction of Apoptosis 1) 2 x 10 4 cells/50 μ L of Jurkat cells or WR19L12a cells (human Fas transfectant) are cultured in 96-well microplate at 37 o C in 5 % CO 2 incubator with RPMI 1640 containing 10 % fetal calf serum. 2) Add 50 μ L of 200 ng/mL anti-human Fas monoclonal antibody diluted with RPMI 1640 containing 10 % fetal calf serum. 3) Culture for appropriate times at 37 o C in 5 % CO 2 incubator with RPMI 1640 containing 10 % fetal calf serum.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Format: Liquid

Buffer: PBS containing 50 % glycerol, pH 7.2. No preservative is contained.

Konservierungsmittel: Azide free

Lagerung: -20 °C

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

Details zu Caspase 9

Target: Caspase 9 (CASP9) (Caspase 9, Apoptosis-Related Cysteine Peptidase (CASP9))

Andere Bezeichnung: caspase-9

Hintergrund: Apoptosis is a major form of cell death characterized by severa l morphological features that include chromatin condensation and fragmentation, cell membrane blebbing, and formation of apoptotic bodies. These morphological changes occur via signaling pathway that leads to the recruitment and activation of caspases, a family of cysteine-containing , aspartate-specific proteases. Caspases exist as inactive proenzymes in cells and are activated through their processing into two subunits in response to apoptotic stimula tion. Activated caspases cleave a variety of important cellular proteins, other caspases, and Bcl-2 family members, leading to a commitment to cell death. Caspase-9, a 45 kDa protein (also known as ICE-LAP6 or Mch-6), is involved in one of relatively well characterized caspase cascades. It is triggered by cytochrome c released from m itochondria, which promotes the activation of caspase-9 by forming a complex with APAF-1 in the presence of dATP. For this activation, physical association of casp ase-9 and APAF-1 in the complex is crucial and it is mediated by the interaction of respective caspase recruitment domain (CARD). This activated caspase-9 induces the downstream caspase-3 activation. It is also reported that the mice lacking caspase-9 die perinatally with a markedly enlarged and malformed cerebrum caused by reduced apoptosis during brain development, and that the thymocyte of the mice show resistance to a subset of apoptotic stimuli.

UniProt: P55211

Pathways: MAPK Signalweg, RTK Signalweg, Apoptose, Caspase Kaskade in der Apoptose, Fc-epsilon Rezeptor Signalübertragung, EGFR Signaling Pathway, Neurotrophin Signalübertragung, Positive Regulation of Endopeptidase Activity