ASK-1 (phospho-S83) antibody

• Catalog Number: orb344610
• Category: Antibodies
• Description: ASK-1 (phospho-S83) antibody
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: ELISA, WB
• Reactivity: Human
• Isotype: IgG
• Immunogen: This purified antibody was prepared from rabbit serum after repeated immunizations with a KLH conjugated peptide corresponding to an internal region near amino acids 75-100 of human ASK-1 protein.
• Concentration: 1.0 mg/mL
• Dilution range: ELISA: 1:5,000 - 1:10,000, WB: 1:1,000
• Form/Appearance: Liquid (sterile filtered)
• Purity: This product is an IgG fraction antibody purified from antiserum by a multi-step process which includes delipidation, salt fractionation and ion exchange chromatography followed by extensive dialysis against the buffer stated above. Assay by immunoelectrophoresis resulted in a single precipitin arc against anti-Rabbit Serum. No reaction was observed with ASK-1 from mouse sources. Reactivity with the kinase from other sources has not been determined.
• Conjugation: Unconjugated
• UniProt ID: Q99683
• NCBI: NP_005914.1
• Storage: Store vial at -20° C prior to opening. Aliquot contents and freeze at -20° C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4° C as an undiluted liquid. Dilute only prior to immediate use.
• Buffer/Preservatives: 0.01% (w/v) Sodium Azide
• Alternative names: rabbit anti-ASK1 pS83 Antibody, Apoptosis signal r
• Note: For research use only
• Application notes: This phospho specific polyclonal antibody reacts human pS83 ASK1 and shows minimal reactivity by western blot, ELISA and competitive ELISA with non-phosphorylated ASK1. Although not tested, this antibody is likely functional in RIA, immunohistochemistry and immunoprecipitation. For immunoblotting a 1:1,000 dilution is recommended. A 155 kDa band corresponding to human ASK-1 is detected. Whole cell lysates from SW1353 can be used as a positive control. For ELISA a 1:5,000 to 1:10,000 dilution is recommended. Researchers should determine optimal titers for other applications.
• Expiration Date: 12 months from date of receipt.

ELISA results of purified polyclonal anti-pS83 ASK-1 antibody tested against BSA conjugates of non-phospho and phospho forms of immunizing peptide. Each well was coated with 0.1 mg of conjugate. The starting dilution of antibody was 1:1000 and each point on the X-axis represents a 2-fold dilution. HRP conjugated Gt-a-Rabbit IgG H&L and TMB substrate were used for detection.

Western blot of anti-pS83 ASK1 antibodies shows specificity for phosphorylated human ASK1. Anti-pS83 (aa 76-87) antibody was tested by western blot against Cos-7 cell lysates after transient transfection with 1) vector only, 2) recombinant HA-ASK1, and 3) recombinant human HA-ASK1 where S83 was substituted with an alanine residue. Cells were lysed 24 h post-transfection in 200 µL of 1x SDS-sample buffer, heated at 96°C for 5', and vortexed for 30 sec. Samples (10 µL each) were separated on a 12% SDS-PAGE gel and transferred to PVDF (Millipore) followed by blocking for 45' using TTBS supplemented with 5% non-fat dry milk. All incubations were performed at room temperature. In panel a) all samples were incubated with anti-HA antibody. This blot demonstrates both recombinant transfections express rASK1. In panel b) all samples were incubated with anti-pS83 ASK1. Lane 2 shows strong specific staining of ASK1. Lane 3, where S83 was replaced with alanine, shows greatly diminished staining. In panel c) all samples were incubated with anti-pS83 ASK1 antibody as before except the antibody was pre-incubated with phospho peptide prior to membrane incubation. No staining is observed after phospho peptide blocking occurs.