Recombinant Human BTK His-tag Protein, CF

Recombinant Human BTK His-tag (Catalog # 11750-BK) is measured by its ability to transfer phosphate from adenosine triphosphate (ATP) to a synthetic peptide substrate.

2 μg/lane of Recombinant Human BTK His-tag Protein (Catalog # 11750-BK) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 82-90 kDa, under reducing conditions.

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human BTK His-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer phosphate from adenosine triphosphate (ATP) to a synthetic peptide substrate.
  The specific activity is >80 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human BTK protein
  

  Met	6-His tag	Sumo-tag (mutated, uncleavable)	3c Protease site	Human BTK (Ala2-Ser659) Accession # Q06187.3

• N-terminal Sequence: Protein identity confirmed by mass spectrometry.
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, 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:
  • Enzyme Activity
• Theoretical MW: 89 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: 82-90 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: >95%, 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 BTK (rhBTK) His-tag (Catalog # 11750-BK)
  • Poly (4:1 Glu:Tyr), 1 mg/mL stock in 25 mM Tris, pH 7.5
  • Adenosine triphosphate (ATP), 10 mM stock in deionized water
  • ADP-Glo Kinase Assay Kit (Promega)
  • White 96-well Plate
  • Plate Reader with Luminescence Read Capability
  1. Dilute rhBTK to 5 µg/mL in Assay Buffer.
  2. Prepare Substrate Mixture containing 200 µM ATP and 0.4 mg/mL Poly (4:1 Glu:Tyr) in Assay Buffer.
  3. Combine equal volumes of 5 µg/mL rhBTK 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 of Kinase Detection Reagent 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:
  • rhBTK: 2.5 µg/mL
  • ATP: 100 µM
  • Poly (4:1 Glu:Tyr): 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 BTK His-tag Protein, CF

• Agammaglobulinaemia tyrosine kinase
• AGMX1
• AGMX1MGC126262
• AT
• ATKIMD1
• B-cell progenitor kinase
• BPK
• Bruton agammaglobulinemia tyrosine kinase
• Bruton tyrosine kinase
• BTK
• dominant-negative kinase-deficient Brutons tyrosine kinase
• EC 2.7.10
• EC 2.7.10.2
• IMD1
• PSCTK1
• tyrosine-protein kinase BTK
• XLA
• XLAMGC126261

Background

Bruton tyrosine kinase (BTK), also known as Agammaglobulinemia tyrosine kinase (ATK) and B-cell progenitor kinase (BPK), is one of five members of the tyrosine kinase expressed in hepatocellular carcinoma (TEC) family of cytoplasmic non-receptor tyrosine kinases (1,2) that is primarily expressed in B-lymphocytes. BTK, like others in this family, contains a characteristic N-terminal pleckstrin homology (PH) domain as well as a TEC homology domain with three regions including a Btk homology region and two proline-rich regions. In addition, BTK contains two Src homology domains that mediate binding events and a C-terminal kinase domain (1) with a conserved catalytic domain with an ATP binding site within the cleft of two lobes at the N-terminal and C-terminal regions (1,2). BTK is activated by phosphorylation by SYK or SRC family kinases and then subsequent autophosphorylation after recruitment to the membrane through its interaction with PIP3 production resulting from B cell antigen receptor activation (2). Mutations in the human btk gene were found to cause X-linked agammaglobulinemia (XLA), a male immune deficiency disorder characterized by a lack of mature, immunoglobulin-producing, peripheral B cells (3,4). Its activation is known to be involved in signal transduction pathways regulating survival, activation, proliferation, and also differentiation of B lineage lymphoid cells (1, 3-5).  Consequently BTK is critical for the survival of leukemic cells in various B cell malignancies including chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL)(2,6,7). Inhibition of BTK is a promising target for therapeutics for B-cell malignancies given initial small-molecule inhibitors showed excellent anti-tumor activity in clinical studies (8) and their success in therapeutic application for CLL and small lymphocytic leukemia (SLL)(9).  Significant interest remains in the development of higher specificity BTK inhibitors with less off-target activity (10,11) as well as combination inhibitors targeting other kinases or other proteins targets (2,12-14) in addition to BTK in B-cell malignancy. In addition, ectopic expression of BTK has been observed in various solid tumors making BTK a therapeutic target of interest in a broader context for the role the kinase may play in the tumor microenvironment (2,15).

1. Mao, C. et. al. (2001) J. Biol. Chem. 276:41435.
2. Singh, S.P. et. al. (2018) Mol. Cancer 17:57. 
3. Tsukada, S. et. al. (1993) Cell. 72:279.
4. Vetrie, D. et. al. (1993) Nature. 361:226.
5. Kurosaki, T. and M. Kurosaki (1997) J. Biol. Chem. 272:15595.
6. Herman, S.E. et. al. (2011) Blood. 117:6287.
7. Chang, B.Y. et. al. (2013) Blood 122:2412.
8. Byrd, J.C. et. al. (2013) N. Engl. J. Med. 369:32.
9. Burger, J.A. et. al. (2015) N. Engl. J. Med. 373:2425.
10. Wu, J.  et. al. (2016) J. Hematol. Oncol. 9:80.
11. Tan, S. et. al.  (2025) Cancer. 131:e70083.
12. Sagiv-Barfi, I. et. al. (2015) Proc. Natl. Acad. Sci. U.S.A. 112:E966.
13. Yahiaoui, A. et. al. (2017) PLoS One. 12:e0171221.
14. Secchiero, P. et. al. (2017) Oncotarget. 8:59235.
15. Grassilli, E. et. al. (2022) Front. Oncol. 12:944538.