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Mitogen-Activated Protein Kinase Kinase Kinase 5 (MAP3K5) (AA 76-87), (pSer83) antibody

Details for Product No. ABIN116930
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Antigen
Synonyms 7420452D20Rik, ASK, ASK1, MAPKKK5, Mekk5, mitogen-activated protein kinase kinase kinase 5, MUD21.11, MUD21_11, MEKK5, MAP3K5
Epitope
AA 76-87, pSer83
(46), (38), (33), (16), (12), (12), (12), (12), (10), (7), (7), (3), (3), (2), (2), (2), (2), (2), (2), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1)
Reactivity
Human
(309), (129), (76), (36), (24), (12), (1)
Host
Rabbit
(292), (19)
Clonality
Polyclonal
Conjugate
Un-conjugated
(10), (10), (10), (6), (6), (6), (6), (6), (6), (6), (6), (4), (4), (4)
Application
Western Blotting (WB)
(225), (118), (108), (60), (59), (31), (29), (7), (5), (4), (3), (3), (1), (1), (1)
Pubmed 14 references available
Quantity 0.2 mg
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Catalog No. ABIN116930
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Immunogen This purified antibody was prepared from rabbit serum after repeated immunizations with a KLH conjugated peptide corresponding to amino acids 76-87 of human ASK-1 protein.
Specificity 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
Purification Multi-step process.
Alternative Name MEKK5 / ASK1
Background ASK-1 (apoptosis signal-regulating kinase 1- also referred to as MEK Kinase-5 or MAPKKK5) is a novel serine/threonine MAP kinase kinase kinase (MAPKKK) component of the mitogen -activated protein (MAP) cascade that is activated in response to extracellular stimuli by cytokines, growth factors and environmental stresses and other factors. Overexpression of ASK-1 induces apoptotic cell death. ASK-1 is expressed in a variety of human and mouse tissues. The overall amino acid sequence identity between the mouse and human ASK1 is 91.9%. ASK-1 interacts with CDKN1A (also known as p21, WAF1, CIP1). Please refer to the reference list at the end of this document for further information.
Alternate names: Apoptosis signal-regulating kinase 1, MAP3K5, MAPK/ERK kinase kinase 5, MAPKKK5, Mitogen-activated protein kinase kinase kinase 5
Gene ID 4217
NCBI Accession NP_005914.1
UniProt Q99683
Application Notes This phospho specific polyclonal antibody reacts human pS83 ASK1 and shows minimalreactivity by Western blot, ELISA and competitive ELISA with non-phosphorylated ASK1. Although not tested, this antibody is likely functional in RIA, Immunohistochemistry andImmunoprecipitation. Recommended Dilutions: For immunoblotting a 1/1,000 dilution is recommended. A 155kDa band corresponding to human ASK-1 is detected. Whole cell lysates from SW1353 canbe used as a positive control. For ELISA a 1/5,000-1/10,000 dilution is recommended. Other applications not tested. Optimal dilutions are dependent on conditions and should be determined by the user.
Restrictions For Research Use only
Format Liquid
Concentration 2.0 mg/mL (by UV absorbance at 280 nm)
Buffer 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2 with 0.01% sodium azide as preservative.
Preservative Sodium azide
Precaution of Use This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handling Advice Dilute only prior to immediate use . Avoid cycles of freezing and thawing.
Storage -20 °C
Storage Comment Store the antibody (in aliquots) at -20°C.
Expiry Date 12 months
Supplier Images
anti-Mitogen-Activated Protein Kinase Kinase Kinase 5 (MAP3K5) (AA 76-87), (pSer83) antibody Immunoblot of anti-pS83 ASK1 antibodies shows specificity for phosphorylated human ASK1. Anti-pS83 (aa 76-87) antibody, generated by immunization with phospho peptide coupled to KLH, was tested by immunoblot against lysates of Cos-7 cells after transient transfection, separately, 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 uL of 1x SDS-sample buffer, heated at 96°C for 5', and vortexed for 30 sec. Samples (10 uL 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 10 ug/mL mouse anti-HA antibody for 45'. After 5X washes with TTBS, reaction with ALP rabbit anti-mouse IgG at 200 ng/mL proceeded for 45' following again by washing as before. The blot was developed using BCIP/NBT. This blot demonstrates both recombinant transfections were successfully over-expressed in the Cos-7 cells. In panel b) all samples were incubated with a 1:1,000 dilution of Rockland's anti-pS83 ASK1 antibody for 45'. The antibody was pre-incubated with non-phospho peptide prior to membrane incubation. After 5X washes with TTBS, reaction with HRP goat anti-rabbit IgG at 10 ng/mL proceeded for 45' following again by washing as before. The membrane was processed as before. 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 a 1:1,000 dilution of Rockland's 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.
anti-Mitogen-Activated Protein Kinase Kinase Kinase 5 (MAP3K5) (AA 76-87), (pSer83) antibody (2) ELISA results of purified polyclonal anti-pS83 ASK-1 (aa 76-87) 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 1:1,000 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.
Background publications Kanamoto, Mota, Takeda et al.: "Role of apoptosis signal-regulating kinase in regulation of the c-Jun N-terminal kinase pathway and apoptosis in sympathetic neurons." in: Molecular and cellular biology, Vol. 20, Issue 1, pp. 196-204, 2000 (PubMed).

Yasuda, Inoue, Hirabayashi et al.: "Triggering of neuronal cell death by accumulation of activated SEK1 on nuclear polyglutamine aggregations in PML bodies." in: Genes to cells : devoted to molecular & cellular mechanisms, Vol. 4, Issue 12, pp. 743-56, 2000 (PubMed).

Liu, Nishitoh, Ichijo et al.: "Activation of apoptosis signal-regulating kinase 1 (ASK1) by tumor necrosis factor receptor-associated factor 2 requires prior dissociation of the ASK1 inhibitor thioredoxin." in: Molecular and cellular biology, Vol. 20, Issue 6, pp. 2198-208, 2000 (PubMed).

Takeda, Hatai, Hamazaki et al.: "Apoptosis signal-regulating kinase 1 (ASK1) induces neuronal differentiation and survival of PC12 cells." in: The Journal of biological chemistry, Vol. 275, Issue 13, pp. 9805-13, 2000 (PubMed).

Hatai, Matsuzawa, Inoshita et al.: "Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation." in: The Journal of biological chemistry, Vol. 275, Issue 34, pp. 26576-81, 2000 (PubMed).

Mochida, Takeda, Saitoh et al.: "ASK1 inhibits interleukin-1-induced NF-kappa B activity through disruption of TRAF6-TAK1 interaction." in: The Journal of biological chemistry, Vol. 275, Issue 42, pp. 32747-52, 2000 (PubMed).

Sayama, Hanakawa, Shirakata et al.: "Apoptosis signal-regulating kinase 1 (ASK1) is an intracellular inducer of keratinocyte differentiation." in: The Journal of biological chemistry, Vol. 276, Issue 2, pp. 999-1004, 2001 (PubMed).

Noguchi, Kokubu, Kitanaka et al.: "ASK1-signaling promotes c-Myc protein stability during apoptosis." in: Biochemical and biophysical research communications, Vol. 281, Issue 5, pp. 1313-20, 2001 (PubMed).

Tobiume, Matsuzawa, Takahashi et al.: "ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis." in: EMBO reports, Vol. 2, Issue 3, pp. 222-8, 2001 (PubMed).

Cho, Lee, Park et al.: "Glutathione S-transferase mu modulates the stress-activated signals by suppressing apoptosis signal-regulating kinase 1." in: The Journal of biological chemistry, Vol. 276, Issue 16, pp. 12749-55, 2001 (PubMed).

Geleziunas, Xu, Takeda et al.: "HIV-1 Nef inhibits ASK1-dependent death signalling providing a potential mechanism for protecting the infected host cell." in: Nature, Vol. 410, Issue 6830, pp. 834-8, 2001 (PubMed).

Zou, Tsutsui, Ray et al.: "The cell cycle-regulatory CDC25A phosphatase inhibits apoptosis signal-regulating kinase 1." in: Molecular and cellular biology, Vol. 21, Issue 14, pp. 4818-28, 2001 (PubMed).

Matsuzawa, Ichijo: "Molecular mechanisms of the decision between life and death: regulation of apoptosis by apoptosis signal-regulating kinase 1." in: Journal of biochemistry, Vol. 130, Issue 1, pp. 1-8, 2001 (PubMed).

Morita, Saitoh, Tobiume et al.: "Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress." in: The EMBO journal, Vol. 20, Issue 21, pp. 6028-36, 2001 (PubMed).

Validation Images
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