Product Pathways - Kinases
Btk Kinase #7377
Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.
Description
Purified recombinant full-length human Btk (Met1-Ser659) kinase, supplied as a protein with amino-terminal His tag.
Source / Purification
The His tag protein was produced using a baculovirus expression system with a construct expressing full-length human Btk (Met1-Ser659) (GenBank Accession No. NM_000061) with an amino-terminal His tag. The protein was purified by Immobilized Metal Affinity Chromatography (IMAC).
Gel Staining
Figure 1. The purity of the Btk fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. Btk kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: Poly EY (E:Y, 4:1) 200 ng/μL, and recombinant Btk: variable.
Quality Control
The theoretical molecular weight of the Btk fusion protein is 78 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. Btk kinase activity was determined using a radiometric assay [Fig.2].
Background
Bruton's tyrosine kinase (Btk) is a member of the Btk/Tec family of cytoplasmic tyrosine kinases. Like other Btk family members, it contains a pleckstrin homology (PH) domain, and Src homology SH3 and SH2 domains. Btk plays an important role in B cell development (1,2). Activation of B cells by various ligands is accompanied by Btk membrane translocation mediated by its PH domain binding to phosphatidylinositol-3,4,5-trisphosphate (3-5). The membrane-located Btk is active and associated with transient phosphorylation of two tyrosine residues, Tyr551 and Tyr223. Tyr551 in the activation loop is transphosphorylated by the Src family tyrosine kinase, leading to autophosphorylation at Tyr223 within the SH3 domain, which is necessary for full activation (6,7). The activation of Btk is negatively regulated by PKCβ through phosphorylation of Btk at Ser180, which results in reduced membrane recruitment, transphosphorylation and subsequent activation (8). The PKC inhibitory signal is likely to be a key determinant of the B-cell receptor signaling threshold to maintain optimal Btk activity (8).
- Khan, W.N. (2001) Immunol. Res. 23, 147-156.
- Lewis, C.M. et al. (2001) Curr. Opin. Immunol. 13, 317-325.
- Salim, K. et al. (1996) EMBO J. 15, 6241-6250.
- Rameh, L.E. et al. (1997) J. Biol. Chem. 272, 22059-22066.
- Varnai, P. et al. (1999) J. Biol. Chem. 274, 10983-10989.
- Rawlings, D.J. et al. (1996) Science 271, 822-825.
- Park, H. et al. (1996) Immunity 4, 515-525.
- Kang, S.W. et al. (2001) EMBO J. 20, 5692-5702.
Application References
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Companion Products
- 9805 HTScan® Tyrosine Kinase Buffer (4X)
- 9804 ATP (10 mM)
- 7450 Tyrosine Kinase Substrate Screening Kit
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
