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BD Pharmingen™ Bioimaging Certified Reagent Technical Data Sheet Polyclonal Rabbit Anti-Human Caspase-8 Product Information Material Number: 551234 Alternate Name: FLICE, MACH-1, Mch5 Reactivity: QC Testing: Human Component: Description: Size: Immunogen: Target MW: Storage Buffer: 51-9000095 Polyclonal Rabbit Anti-Human Caspase-8 (FLICE, MACH-1, Mch5) Component: Description: Size: Concentration: Storage Buffer: 51-16526N Jurkat Cell Lysate 100 µl (1 ea) Human Caspase-8 aa 2-20 Peptide 50/55 kD, 36/40 kD, and 23 kD Aqueous buffered solution containing ≤0.09% sodium azide. 50 µg (1 ea) 1.0 mg/ml 1 mg/ml in SDS-PAGE buffer (62mM Tris pH 6.8, 2% SDS, 0.9% b-mercaptoethanol, 0.003% bromophenol blue, 5% glycerol) Description Caspase-8 is a cytosolic protein with homology to the CD95/Fas-associated signal transducer, FADD/MORT-1, as well as to other caspase (ICE/Ced-3) cysteine proteases. Caspase-8 is produced as a proenzyme which upon receptor aggregation is proteolytically cleaved into smaller subunits of 40/36 kDa (doublet) and 23 kDa subunits. These subunits form a proteolytically active heterodimer capable of cleaving other caspase family members, as well as substrates such as PARP. This polyclonal antibody recognizes both the proform of caspase-8 as a 55/50 kDa doublet as well as the cleaved forms which migrate at 40/36 kDa (doublet) and 23 kDa in SDS/PAGE. This antibody is routinely tested by Western blot analysis and immunofluorescent imaging. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature. Preparation and Storage The polyclonal antibody was purified from antiserum by negative adsorption and affinity chromatography. Store the polyclonal antibody undiluted at 4°C. Store the Jurkat cell lysate undiluted at -20°C. Application Notes Application Bioimaging Routinely Tested Western blot Routinely Tested Recommended Assay Procedure: Methanol Procedure for a 96 well plate: Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 90% methanol. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Wash three times with PBS. Flick out PBS and add second step reagent. Incubate for 1 hour at RT. Wash three times with PBS. Image sample. Triton-X 100 Procedure for a 96 well plate: Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 0.1% Triton-X 100. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Flick out and wash three times with PBS. Flick out and add second step reagent. Incubate for 1 hour at RT. Flick out and wash three times with PBS. Image sample. 551234 Rev. 5 Page 1 of 2 Western blot analysis of caspase-8. Lysates from control (lane 1) or apoptotic (lane 2) Jurkat cells were probed with the anti-caspase-8 antibody at a dilution of 1:3000. The proform of caspase-8 migrates at ~55/50 kDa (doublet), while the cleaved forms migrate at ~40/36 (doublet) and ~23 kDa. Immunofluorescent staining of U2OS cells. Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10,000 cells per well. After overnight incubation, cells were stained using the Triton X100 fix/perm protocol (see Recommended Assay Procedure) and the anti- caspase-8 antibody. The second step reagent was FITC goat antirabbit Ig (Cat. No. 554020). Images were taken on a Pathway 850 imager using a 20x objective. This antibody also stained HeLa and A549 cells and worked with both the Triton X100 and Methanol fix/perm protocols (see Recommended Assay Procedure). Suggested Companion Products Catalog Number Name Size Clone 554021 HRP Goat Anti-Rabbit Ig 1.0 ml (none) 554020 FITC Goat Anti-Rabbit Ig 0.5 mg (none) 550959 Jurkat Apoptotic Lysate Set I 500 µg (none) 611451 Jurkat Cell Lysate 500 µg (none) Product Notices 1. Since applications vary, each investigator should titrate the reagent to obtain optimal results. 2. Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols. 3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing. References Boesen-de Cock JG, Tepper AD, de Vries E, van Blitterswijk WJ, Borst J. Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation. J Biol Chem. 1999; 274(20):14255-14261.(Biology: Western blot) Cock JG, Tepper AD, de Vries E, van Blitterswijk WJ, Borst J. CD95 (Fas/APO-1) induces ceramide formation and apoptosis in the absence of a functional acid sphingomyelinase. J Biol Chem. 1998; 273(13):7560-7565.(Immunogen: Western blot) Muzio M, Chinnaiyan AM, Kischkel FC, et al. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death--inducing signaling complex. Cell. 1996; 85(6):817-827.(Biology) 551234 Rev. 5 Page 2 of 2