Cell Signaling Technology

Product Pathways - Lymphocyte Signaling

Phospho-Btk (Tyr223) Antibody #5082

Applications Reactivity Sensitivity MW (kDa) Source
W H (M) (R) Endogenous 77 Rabbit

Applications Key:  W=Western Blotting
Reactivity Key:  H=Human  M=Mouse  R=Rat
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Phospho-Btk (Tyr223) Antibody detects endogenous levels of Btk only when phosphorylated at Tyr223.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr223 of human Btk protein. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from Ramos cells, untreated or treated with anti-human IgM (12 µg/ml for 10 minutes), using Phospho-Btk (Tyr223) Antibody (upper) or Btk (C82B8) Rabbit mAb #3533 (lower).

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-localized 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 kinases, 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).

  1. Khan, W.N. (2001) Immunol. Res. 23, 147-156.
  2. Lewis, C.M. et al. (2001) Curr. Opin. Immunol. 13, 317-325.
  3. Salim, K. et al. (1996) EMBO J. 15, 6241-6250.
  4. Rameh, L.E. et al. (1997) J. Biol. Chem. 272, 22059-22066.
  5. Varnai, P. et al. (1999) J. Biol. Chem. 274, 10983-10989.
  6. Rawlings, D.J. et al. (1996) Science 271, 822-825.
  7. Park, H. et al. (1996) Immunity 4, 515-525.
  8. Kang, S.W. et al. (2001) EMBO J. 20, 5692-5702.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

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