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Recombinant Mouse Active JNK1 Protein, CF

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The approximate molecular weight is 70 kDa and the average purity is 83%.
The approximate molecular weight is 70 kDa and the average purity is 83%.

Product Details

Summary
Reactivity MuSpecies Glossary
Applications Bioactivity
Format
Carrier-Free

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Recombinant Mouse Active JNK1 Protein, CF Summary

Details of Functionality
The specific activity of JNK1 was determined to be 104 nmol/min/mg using an ATF2 substrate and was equivalent to 122 nmol/min/mg as per radiometric assay.
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived mouse JNK1 protein
Accession #
N-terminal Sequence
Using an N terminal GST tag
Protein/Peptide Type
Recombinant Proteins
Gene
Mapk9
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Applications/Dilutions

Dilutions
  • Bioactivity
SDS-PAGE
70 kDa
Publications
Read Publication using 1776-KS.

Packaging, Storage & Formulations

Storage
This product is stable at ≤ ‑70°C for up to 1 year from the date of receipt. For optimal storage, aliquot into smaller quantities after centrifugation and store at recommended temperature. Avoid repeated freeze-thaw cycles.
Buffer
Supplied in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM Glutathione, 0.25 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol.
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
Assay Procedure
  • Active Kinase - Active JNK1 (0.1 μg/μL) diluted with Kinase Dilution Buffer IX and assayed as outlined in sample activity plot. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
  • Kinase Assay Buffer III (5X) - 200 mM Tris-HCl, pH 7.4, 100 mM MgCl2, and 0.5 mg/mL BSA. Add fresh DTT prior to use to a final concentration of 250 μM.
  • Kinase Dilution Buffer IX (1X) - Kinase Assay Buffer III diluted at a 1:4 ratio (5X dilution) with cold, distilled water. Add fresh DTT prior to use to a final concentration of 50 μM.
  • ADP-GloTM Kinase Assay Kit - 10 mM of ATP Solution, 10 mM of ADP Solution, ADP-GloTM Reagent, and Kinase Detection Reagent.
  • Substrate - ATF2 substrate prepared in buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM Glutathione, 0.1 mM EDTA, and 0.25 mM DTT) to a final concentration of 0.2 mg/mL.
  1. Thaw the Active JNK1, Kinase Assay Buffer III (5X), and Substrate on ice. Prepare a 15 μL enzyme dilution using Kinase Dilution Buffer IX (1X), at the desired concentration, in a pre-chilled 96-well plate.
  2. Prepare a Substrate/ATP mixture as follows (25 μM example):
    a. 10 mM ATP Solution: 1 μL
    b. Kinase Assay Buffer III (5X): 79 μL
    c. Substrate at 0.2 mg/mL: 80 μL
  3. Transfer the following reaction components prepared in Steps 1 and 2 to a 384-well opaque plate, bringing the reaction volume up to 5 μL:
    a. 3 μL of diluted Active JNK1
    b. 2 μL of Substrate/ATP mix as prepared in Step 2. This initiates the reaction.
  4. Set up the blank control as outlined in Step 2, excluding the addition of the kinase. Replace the kinase with an equal volume of Kinase Dilution Buffer IX (1X).
  5. Incubate at ambient temperature for 40 minutes.
  6. After the 40 minute incubation period, terminate the reaction and deplete the remaining ATP by adding 5 μL of ADP-Glo Reagent. Spin down and shake the 384-well plate. Then incubate the reaction mixture for another 40 minutes at ambient temperature.
  7. Add 10 μL of the Kinase Detection Reagent to the 384-well plate and incubate the reaction mixture for another 30 minutes at ambient temperature.
  8. Read the 384-well reaction plate using the Luminescence Module Protocol on a GloMax®-Multi Microplate Multimode Reader.
  9. Determine the corrected activity (RLU) by removing the blank control value (see step 4) for each sample and calculate the kinase specific activity as outlined below.


    Calculation of Specific Activity of ADP (RLU/pmol)
    From ATP-ADP conversion vurce, determine RLU/pmol of ADP

    Kinase Specific Activity (SA) (pmol/minutes/μg or nmol/minutes/mg)
    Corrected RLU from reaction / [(SA of ADP in RLU/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)]

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Active JNK1 Protein, CF

  • c-Jun N-terminal kinase 1
  • EC 2.7.11
  • EC 2.7.11.24
  • JNK1 alpha protein kinase
  • JNK1 beta protein kinase
  • JNK1
  • JNK1JNK1A2
  • JNK-46
  • JUN N-terminal kinase
  • MAP kinase 8
  • MAPK 8
  • MAPK8
  • mitogen-activated protein kinase 8 isoform JNK1 alpha1
  • mitogen-activated protein kinase 8 isoform JNK1 beta2
  • mitogen-activated protein kinase 8
  • PRKM8
  • PRKM8JNK
  • protein kinase JNK1
  • SAPK1
  • SAPK1JNK21B1/2
  • Stress-activated protein kinase 1
  • Stress-activated protein kinase JNK1

Background

JNK1 is a member of the MAP kinase group that is activated by dual phosphorylation on Threonine and Tyrosine residues during exposure to stress such as UV irradiation. JNK1 binds to the c-Jun transactivation domain and phosphorylates it on S63 and S73 (1). JNK1 has been shown to play an important role in disease processes. Activation of JNK1 results in defects in myotube viability and integrity leading to dystrophic myofiber destruction (2). JNK1 activity is also abnormally elevated in obesity and removal of JNK1 results in decreased adiposity and significantly improved insulin sensitivity.
  1. Derijard, B. et al. (1994) Cell 76:1025.
  2. Kolodziejczyk, S.M. et al. (2001) Curr. Biol. 11:1278.

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Bioinformatics

Gene Symbol Mapk9
Uniprot