A part of MilliporeSigma

17-466 | phospho-JNK 1/2 (Thr183/Tyr185) STAR ELISA Kit

17-466
96 assays  
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      Overview

      Replacement Information

      Key Specifications Table

      Analytes AvailableSpecies ReactivityKey ApplicationsDetection Methods
      JNK 1/2 H, M ELISA Chromogenic
      Description
      Catalogue Number17-466
      Brand Family Upstate
      Trade Name
      • STAR
      • Upstate
      Descriptionphospho-JNK 1/2 (Thr183/Tyr185) STAR ELISA Kit
      Background InformationTest Principle
      The UPSTATE colorimetric STAR (Signal Transduction Assay Reaction) ELISA kit is a solid phase sandwich enzyme linked immunosorbent assay that provides a fast, sensitive method to detect specific levels of signaling targets in denatured cell extracts. The JNK 1/2 plate is coated with a specific mouse monoclonal JNK 1/2 capture antibody on the microwells of the 96-well clear plate. Sample lysate or the standard included in the kit are incubated in the microwells allowing JNK 1/2 antigen to be captured in the plate wells. The plate is then washed to remove any non-bound unspecific material. The wells are then incubated with a specific rabbit anti-phospho-JNK (Thr183/Tyr185) antibody to detect the captured JNK on the plate well that has been phosphorylated on Thr183 and Tyr185. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. This allows for a sensitive enzymatic detection of the sample. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader. The entire assay takes less than 5 hours to complete with minimal hands-on time. Many of the reagents are supplied in ready-to use formulations for ease of use. The kit also includes a standard that is run as both a positive control and to develop a standard line of detection.

      JNK Background
      JNK (c-Jun N-terminal Kinase, MAPK8) is a member of the MAP kinase family. JNK was originally found to be stress activated, and as such are also referred to as the stress-related MAPK. Four alternatively spliced transcript variants encoding distinct isoforms of JNK have been reported. JNK is regulated by stress inducing signals such as osmotic stress and UV radiation as well as proinflammatory cytokines such as IL-1 and TNFα. Just as Erk (p44/p42, MAPK) is activated by its upstream kinase MEK, JNK is phosphorylated and activated by MKK4 and MKK7 on the quintessential MAPK activation sequence of Thr-X-Tyr, where X is a Pro for the JNK kinases on Thr183/Tyr185 for JNK1 and Thr221/Tyr223 for JNK2. Once phosphorylated and activated, JNK goes on to phosphorylate downstream targets in both the cytoplasm and the nucleus. JNK serves as a transcriptional regulator and is known to phosphorylate a number of downstream targets, including the transcription factors c-Jun, ATF-2, and ELK-1. C-Jun, a member of the AP-1 family of transcription factors, regulates cytokine gene expression. Along these lines, JNK inhibitors may be effective in the control of rheumatoid arthritis (RH), an autoimmune disease involving increased production of inflammatory cytokines. Inhibition of JNK signaling also enhances chemotherapy-induced inhibition of tumor cell growth, suggesting the JNKs may provide a molecular target for the treatment of cancer. Both JNK and p38 MAPK are often simultaneously activated in response to environmental changes. These two stress kinase signaling pathways have evolved to relay increasingly complex ranges of environmental stimuli. The JNK pathway is critically involved in diabetes and levels are abnormally elevated in obesity. The cell-permeable JNK inhibitory peptide may have promise as a therapeutic agent for diabetes.
      Materials Required but Not Delivered1. Multi-channel or repeating pipettes
      2. Plate shaker (optional)
      3. Pipettors & tips capable of accurately measuring
      1-1000μL
      4. Graduated serological pipettes
      5. 96-well microtiter Plate Reader with 450 nm filter
      6. Graphing software for plotting data or graph paper for manual
      plotting of data
      7. Microfuge tubes for standard and sample dilutions
      8. Mechanical vortex
      9. 1 liter container
      10. Distilled or deionized water
      References
      Product Information
      Components
      • 1. Capture Plate pre-coated with anti-JNK1/2 antibody: (Part No. 17-466A) One pre-coated 96-stripwell immunoplate sealed in a foil pouch.
      • 2. Anti-phospho-JNK (Thr183/Tyr185) detection antibody: (Part No. 17-466B) One bottle (11 mL) of anti- phospho- JNK (Thr183/Tyr185) detection antibody containing sodium azide, ready to use.
      • 3. ELISA Diluent: (Part No. 17-466C) One bottle (25 mL) of ELISA Diluent containing sodium azide, ready to use.
      • 4. 25X ELISA Wash Buffer: (Part No. 17-466D) One bottle (50 mL) of 25X ELISA Wash Buffer.
      • 5. Anti-Rabbit IgG HRP conjugate: (Part No. 17-466E) One vial (125 µL) of 100X anti-rabbit HRP conjugate containing thimerosal.
      • 6. HRP Diluent: (Part No. 17-466F) One bottle (25 mL) of HRP Diluent containing thimerosal.
      • 7. TMB Solution: (Part No. 17-466G) One bottle (25 mL) of stabilized tetramethylbenzidine (TMB), ready to use.
      • 8. Stop Solution: (Part No. 17-466H) One bottle (25 mL) of stop solution, ready to use.
      • 9. Phospho- JNK (Thr183/Tyr185) Standard: (Part No. 17-466I) Two vials of phosphorylated JNK (Thr183/Tyr185) standard, lyophilized.
      • 10. Plate Covers: Two plate covers.
      Detection methodChromogenic
      Applications
      ApplicationThe phospho-JNK 1/2 (Thr183/Tyr185) colorimetric STAR ELISA kit is a solid phase sandwich enzyme linked immunosorbent assay that provides a fast, sensitive method to quantify specific levels of signaling targets in denatured cell extracts.
      Key Applications
      • ELISA
      Biological Information
      Species Reactivity
      • Human
      • Mouse
      Analytes Available
      • JNK 1/2
      Entrez Gene Number
      Entrez Gene SummaryThe protein encoded by this gene is a member of the MAP kinase family. MAP kinases act as an integration point for multiple biochemical signals, and are involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. This kinase targets specific transcription factors, and thus mediates immediate-early gene expression in response to various cell stimuli. It is most closely related to MAPK8, both of which are involved in UV radiation induced apoptosis, thought to be related to the cytochrome c-mediated cell death pathway. This gene and MAPK8 are also known as c-Jun N-terminal kinases. This kinase blocks the ubiquitination of tumor suppressor p53, and thus it increases the stability of p53 in nonstressed cells. Studies of this gene's mouse counterpart suggest a key role in T-cell differentiation. Four alternatively spliced transcript variants encoding distinct isoforms have been reported.
      Gene Symbol
      • MAPK8
      • JNK1
      • JNK21B1/2
      • PRKM8
      • JNK-46
      • JNK
      • SAPK1
      • JNK1A2
      Modifications
      • Phosphorylation
      UniProt Number
      UniProt SummaryFUNCTION: SwissProt: P45984 # JNK2 isoforms display different binding patterns: alpha- 1 and alpha-2 preferentially bind to c-Jun, whereas beta-1 and beta-2 bind to ATF2. However, there is no correlation between binding and phosphorylation, which is achieved at about the same efficiency by all isoforms. JUNB is not a substrate for JNK2 alpha-2, and JUND binds only weakly to it.
      COFACTOR: Magnesium.
      SIZE: 424 amino acids; 48139 Da
      SUBUNIT: Binds to at least four scaffolding proteins, MAPK8IP1/JIP-1, MAPK8IP2/JIP-2, MAPK8IP3/JIP-3/JSAP1 and SPAG9/MAPK8IP4/JIP-4. These proteins also bind other components of the JNK signaling pathway.
      DOMAIN: SwissProt: P45984 The TXY motif contains the threonine and tyrosine residues whose phosphorylation activates the MAP kinases.
      PTM: Dually phosphorylated on Thr-183 and Tyr-185, which activates the enzyme. Autophosphorylated in vitro.
      SIMILARITY: Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. MAP kinase subfamily. & Contains 1 protein kinase domain.
      Physicochemical Information
      Sensitivity
      • Sensitivity: 0.8 unit/mL.
        Range of Detection: 1.6 to 100 units/mL
        Species Reactivity: Human and mouse
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Quality AssuranceRoutinely evaluated by ELISA.
      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Storage Conditions1 year at 4°C from date of shipment
      Packaging Information
      Material Size96 assays
      Transport Information
      Supplemental Information
      Specifications

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      Categories

      Life Science Research > Antibodies and Assays > Immunoassays > Enzyme-linked Immunosorbent Assay (ELISA) > Complete ELISA Kits
      Life Science Research > Protein Detection and Quantification > Immunoassays > Enzyme-linked Immunosorbent Assay (ELISA) > Complete ELISA Kits