Death Receptor Antibody Sampler Kit
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Death Receptor Antibody Sampler Kit Store at -20°C 3 n 1 Kit Orders n 877-616-CELL (2355) (9 x 20 µl) [email protected] Support n 877-678-TECH (8324) [email protected] Web n www.cellsignal.com rev. 06/16 #8356 For Research Use Only. Not For Use In Diagnostic Procedures. Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 Products Included Product # Quantity Mol. Wt. Isotype mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% Fas (C18C12) Rabbit mAb 4233 20 µl 40-50 kDa Rabbit IgG sodium azide. Store at –20°C. Do not aliquot the antibodies. TNF-R1 (C25C1) Rabbit mAb 3736 20 µl 55 kDa Rabbit IgG Recommended Antibody Dilutions: Western blotting 1:1000 TNF-R2 Antibody 3727 20 µl 75 kDa Rabbit IgG Please visit www.cellsignal.com for validation data DcR2 (D13H4) Rabbit mAb 8049 20 µl 45-60 kDa Rabbit IgG and a complete listing of recommended companion DR5 (D4E9) XP® Rabbit mAb 8074 20 µl 40, 48 kDa Rabbit IgG products. FADD Antibody (Human Specific) 2782 20 µl 28 kDa Rabbit IgG TRADD (7G8) Rabbit mAb 3684 20 µl 32 kDa Rabbit IgG DcR3 Antibody 4758 20 µl 32 kDa Rabbit IgG RIP (D94C12) XP® Rabbit mAb 3493 20 µl 78 kDa Rabbit IgG Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat See www.cellsignal.com for individual component applications, species cross-reactivity, dilutions and additional application protocols. Description: The Death Receptor Antibody Sampler Kit Specificity/Sensitivity: Each antibody in the Death Re- Background References: provides an economical means to investigate the machinery ceptor Antibody Sampler Kit recognizes endogenous levels (1) Nagata, S. (1997) Cell 88, 355-65. of death receptor-mediated apoptosis. The kit includes of the respective target protein. The TNF-R1, DR5, and enough of each primary antibody to perform two western RIP antibodies do not cross-react with other related family (2) Thorburn, A. (2004) Cell Signal 16, 139-44. blot experiments for each primary antibody. members. TNF-R1 (C25C1) Rabbit mAb may recognize a (3) Sheridan, J.P. et al. (1997) Science 277, 818-21. 30 kDa splice isoform of TNF-R1 in some cell lines. Background: The tumor necrosis factor receptor family, (4) Marsters, S.A. et al. (1997) Curr Biol 7, 1003-6. which includes TNF-RI, Fas, DR3, DR4, DR5, and DR6, plays Source/Purification: Monoclonal antibodies are pro- an important role in the regulation of apoptosis in various duced by immunizing animals with synthetic peptides cor- (5) Pitti, R.M. et al. (1998) Nature 396, 699-703. physiological systems (1,2). The receptors are activated by a responding to residues surrounding Lys259 of human Fas (6) Meylan, E. and Tschopp, J. (2005) Trends Biochem Sci family of cytokines that include TNF, FasL, and TRAIL. They protein, Ser331 of human TNF-R1 protein, Arg260 of human 30, 151-9. are characterized by a highly conserved extracellular region DR5 protein, Gly227 of human TRADD protein, Leu190 of (7) Hsu, H. et al. (1996) Immunity 4, 387-96. containing cysteine-rich repeats and a conserved intracellular human RIP protein, or Gly270 of human DcR2 protein. region of about 80 amino acids termed the death domain (8) Stanger, B.Z. et al. (1995) Cell 81, 513-23. Polyclonal antibodies are produced by immunizing animals (DD). The DD is important for transducing the death signal with synthetic peptides corresponding to residues surround- (9) Lin, Y. et al. (1999) Genes Dev 13, 2514-26. by recruiting other DD containing adaptor proteins (FADD, ing Asp335 of human TNF-R2 protein, Ser194 of human TRADD, RIP) to the death-inducing signaling complex (DISC) (10) Ting, A.T. et al. (1996) EMBO J 15, 6189-96. FADD protein, or near the amino terminus of human DcR3 resulting in activation of caspases. Death receptor signaling is (11) Kelliher, M.A. et al. (1998) Immunity 8, 297-303. protein. Polyclonal antibodies are purified by protein A and also controlled by a family of decoy receptors (DcR1, DcR2, peptide affinity chromatography. and DcR3) which lack a cytoplasmic DD and inhibit death re- ceptor-mediated apoptosis by competing for ligand (3-5). The RIP (receptor-interacting protein) family of serine-threonine kinases (RIP, RIP2, RIP3, and RIP4) are important regulators of cellular stress that can trigger pro-survival and inflamma- tory responses through the activation of NF-κB as well as pro-apoptotic pathways (6). In addition to the kinase domain, are trademarks of Cell Signaling Technology, Inc. are trademarks of Cell Signaling Technology, ® RIP contains a death domain responsible for interaction with the death domain receptor Fas and for the recruitment to TNFR1 through interaction with TRADD (6,7). Overexpression of RIP induces both NF-κB activation and apoptosis (7,8). Caspase-8 dependent cleavage of the death domain on RIP can trigger the apoptotic activity of RIP (9). RIP-deficient cells show a failure in TNF-mediated NF-κB activation, making the cells more sensitive to apoptosis (10,11). U.S. Patent No. 5,675,063 Applications Key: W—Western IP—Immunoprecipitation IHC—Immunohistochemistry ChIP—Chromatin Immunoprecipitation IF—Immunofluorescence F—Flow cytometry E-P—ELISA-Peptide and Cell Signaling Technology ® Species Cross-Reactivity Key: H—human M—mouse R—rat Hm—hamster Mk—monkey Mi—mink C—chicken Dm—D. melanogaster X—Xenopus Z—zebrafish B—bovine ® 2014 Cell Signaling Technology, Inc. ® 2014 Cell Signaling Technology, XP Dg—dog Pg—pig Sc—S. cerevisiae All—all species expected Species enclosed in parentheses are predicted to react based on 100% homology. Western Immunoblotting Protocol (Primary Antibody Incubation in BSA) #8356 For Western blots, incubate membrane with diluted antibody in 5% w/v BSA, C Membrane Blocking and Antibody Incubations 1X TBS, 0.1% Tween®20 at 4°C with gentle shaking, overnight. NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized mem- A Solutions and Reagents branes, adjust volumes accordingly. 1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 NOTE: Prepare solutions with Milli-Q or equivalently purified water. minutes at room temperature. 1. 1X Phosphate Buffered Saline (PBS) 2. Incubate membrane in 25 ml of blocking buffer for 1 hour at room temperature. 2. 1X SDS Sample Buffer: 62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 3. Wash three times for 5 minutes each with 15 ml of TBS/T. 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue or phenol red 4. Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml 3. Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol (pH 8.5) primary antibody dilution buffer with gentle agitation overnight at 4°C. 4. 10X Tris Buffered Saline (TBS): To prepare 1 liter of 10X TBS: 24.2 g Tris 5. Wash three times for 5 minutes each with 15 ml of TBS/T. base, 80 g NaCl; adjust pH to 7.6 with HCl (use at 1X). 6. Incubate membrane with HRP-conjugated secondary antibody (1:2000) and 5. Nonfat Dry Milk (weight to volume [w/v]) HRP-conjugated anti-biotin antibody (1:1000) to detect biotinylated protein 6. Blocking Buffer: 1X TBS, 0.1% Tween®20 with 5% w/v nonfat dry milk; for markers in 10 ml of blocking buffer with gentle agitation for 1 hour at room 150 ml, add 15 ml 10X TBS to 135 ml water, mix. Add 7.5 g nonfat dry milk and temperature. mix well. While stirring, add 0.15 ml Tween®20 (100%). 7. Wash three times for 5 minutes each with 15 ml of TBS/T. 7. Wash Buffer: 1X TBS, 0.1% Tween®20 (TBS/T) 8. Bovine Serum Albumin (BSA) D Detection of Proteins 9. Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween®20 with 5% BSA; 1. Incubate membrane with 10 ml LumiGLO® (0.5 ml 20X LumiGLO®, 0.5 ml 20X for 20 ml, add 2 ml 10X TBS to 18 ml water, mix. Add 1.0 g BSA and mix well. Peroxide and 9.0 ml Milli-Q water) with gentle agitation for 1 minute at room While stirring, add 20 µl Tween®20 (100%). temperature. 10. Phototope®-HRP Western Blot Detection System #7071: Includes NOTE: LumiGLO® substrate can be further diluted if signal response is too fast. biotinylated protein ladder, secondary anti-rabbit (#7074) antibody conjugated to horseradish peroxidase (HRP), anti-biotin antibody conjugated to HRP, 2. Drain membrane of excess developing solution (do not let dry), wrap in plastic LumiGLO® chemiluminescent reagent and peroxide. wrap and expose to x-ray film. An initial 10-second exposure should indicate the 11. Prestained Protein Marker, Broad Range (Premixed Format) #7720 proper exposure time. 12. Biotinylated Protein Ladder Detection Pack #7727 NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately 13. Blotting Membrane: This protocol has been optimized for nitrocellulose following LumiGLO® incubation and declines over the following 2 hours. membranes, which CST recommends. PVDF membranes may also be used. B Protein Blotting A general protocol for sample preparation is described below. 1. Treat cells by adding fresh media containing regulator for desired time. 2. Aspirate media from cultures; wash cells with 1X PBS; aspirate. 3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl per plate of 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice. 4. Sonicate for 10–15 seconds to shear DNA and reduce sample viscosity.