Recombinant Human JAK1 kinase domain His-tag Protein, CF

Recombinant Human JAK1 kinase domain His-tag (Catalog # 11738-J1) is measured by its ability to transfer phosphate from adenosine triphosphate (ATP) to a peptide substrate.

2 μg/lane of Recombinant Human JAK1 kinase domain His-tag (Catalog # 11738-J1) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 75-89 kDa, under reducing conditions.

• Reactivity: Hu
• Applications: Bioactivity
• Format: Carrier-Free

Recombinant Human JAK1 kinase domain His-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer phosphate from adenosine triphosphate (ATP) to a peptide substrate.
  
  The specific activity is >110 pmol/min/μg, as measured under the described conditions.
• Source: Human embryonic kidney cell, HEK293-derived human Jak1 protein
  

  Met	6-His tag	Sumo-tag (mutated, uncleavable)	3c Protease site	Human JAK1 (Ala561-Lys1154) Accession # P23458.2

• N-terminal Sequence: Protein identity confirmed by mass spectrometry.
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Bioactivity
• Theoretical MW: 81 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 75-89 kDa, under reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl, DTT and Glycerol.
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 20 mM MgCl₂, 5 mM MnCl₂, 0.1 mg/mL BSA, pH 7.5
  • Recombinant Human JAK1 kinase domain His-tag (rhJAK1) (Catalog # 11738-J1)
  • IRS-1 (TYR608) peptide, 1 mg/mL stock in deionized water
  • Adenosine triphosphate (ATP), 10 mM stock in deionized water
  • ADP-Glo^TM Kinase Assay (Promega)
  • White 96-well Plate
  • Plate Reader with Luminescence Read Capability
  1. Dilute rhJAK1 to 5 µg/mL in Assay Buffer.
  2. Prepare Substrate Mixture containing 200 µM ATP and 0.4 mg/mL IRS-1 (TYR608) peptide in Assay Buffer.
  3. Combine equal volumes of 5 µg/mL rhJAK1 and Substrate Mixture. Create a Substrate Control by replacing enzyme with Assay Buffer.
  4. Incubate at room temperature for 40 minutes in the dark.
  5. After incubation, transfer 10 µL of each reaction to wells of a white plate.
  6. Terminate the reaction and deplete the remaining ATP by adding 10 µL of ADP-Glo Reagent (supplied in kit) to all wells.
  7. Incubate at room temperature for 40 minutes in the dark.
  8. Add 20 µL Kinase Detection Reagent (supplied in kit) to all wells.
  9. Incubate at room temperature for 30 minutes in the dark.
  10. Read plate in Luminescence endpoint mode.
  11. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Luminescence* (RLU) x Conversion Factor** (pmol/RLU)) / (Incubation time (min) x amount of enzyme (µg))

  *Adjusted for Substrate Control
  **Derived from ADP-Glo^TM Kinase Assay Kit protocol (Promega)
  Per Reaction:
  • rhJAK1: 2.5 μg/mL
  • ATP: 100 µM
  • IRS-1 (Tyr608) peptide: 0.2 mg/mL

Notes

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

Alternate Names for Recombinant Human JAK1 kinase domain His-tag Protein, CF

• EC 2.7.10
• Jak1
• JAK-1
• JAK1Atyrosine-protein kinase JAK1
• JAK1B
• Janus kinase 1EC 2.7.10.2
• JTK3

Background

Janus Kinase 1 (JAK1) is one of four members of the JAK, or "just another kinase", family of intracellular, non-receptor tyrosine kinases (1) that noncovalently associate with Type I and Type II cytokine receptor cytoplasmic regions to mediate receptor tyrosine and subsequent STAT protein phosphorylation. JAK1 is broadly expressed in many cell types and contains several conserved JAK homology (JH) domains that compose an N-terminal FERM domain followed by a Src homology (SH) domain that associate and mediate interaction with the cytokine receptors (2,3) and a pseudokinase domain with no catalytic activity that regulates the activity of the catalytic kinase domain present at the C-terminus (4). JAK/STAT signaling is activated by over 50 cytokines that bind to the major cytokine receptors prompting auto-activation of JAKs by transphosphorylation and subsequent phosphorylation of the receptors to promote STAT binding (1,4). Once STAT proteins are bound, JAKs phosphorylate STAT causing dissociation, dimerization, and translocation to the nucleus where they function as transcription factors and play a role in the control of hematopoiesis, the immune response, embryogenesis, inflammation, and cancer (5-8). In addition, JAK1 activating mutations have been directly reported in Castleman's disease (9) and T-cell malignancies such as activating T-ALL, Sezary Syndrome, and T-cell prolymphocytic leukemia (10-12). There is interest in development of JAK inhibitors as a therapeutic target to prevent phosphorylation of their substrates and downstream signaling pathways. Prior clinical studies targeting JAK1 have been focused on rheumatoid arthritis, myelofibrosis, and dermatology-related conditions such as psoriasis (1,13-15). 

1. Agashe, R.P. et al. (2022) Mol. Cancer Ther. 21:1757. 
2. Leonard, W.J. and J.J. O'Shea (1998) Annu. Rev. Immunol. 16:293.
3. Wallweber, H.J. et al. (2014) Nat. Struct. Mol. Biol. 21:443.
4. Garrido-Trigo, A. et al. (2020) J. Crohns Colitis. 14:S713. 
5. Morris, R. et al. (2018) Protein. Sci. 27:1984. 
6. Loh, C.Y. et al. (2019) Front. Oncol. 9:48. 
7. Fragoulis, G.E. et al. (2019) Rheumatology 58:i43. 
8. Brooks, A.J. and T. Putoczki. (2020) Cancers 12:1971.
9. Patel, M. et al. (2017) JAMA Dermatol. 153:449.
10. Kiel, M.J. et. al. (2015) Nat. Commun. 6:8470.
11. Govaerts, I. et al. (2019) Hemasphere 3:e313.
12. Wahnschaffe, L. et. al. (2019) Cancers 11:1833. 
13. Harrington, R. et al. (2020) J. Inflamm. Res. 13:519. 
14. Plosker, G.L. (2015) Drugs 75:297. 
15. Papp, K. et al. (2021) J. Am. Acad. Dermatol. 85:863.