Cell Signaling Technology

Product Pathways - Lymphocyte Signaling

Btk (D3H5) Rabbit mAb #8547

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IHC-P F H M (R) (Hm) (Mk) (B) (Dg) (Pg) (Hr) Endogenous 77 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  B=Bovine  Dg=Dog  Pg=Pig  Hr=Horse
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

* Product-specific protocol.

Specificity / Sensitivity

Btk (D3H5) Rabbit mAb recognizes endogenous levels of total Btk protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp195 of human Btk protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Btk (D3H5) Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Btk (D3H5) Rabbit mAb. Note staining of inflammatory cells.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human B-cell lymphoma using Btk (D3H5) Rabbit mAb.


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded mouse colon using Btk (D3H5) Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Btk (D3H5) Rabbit mAb. Note staining of inflammatory cells.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded cell pellets, Ramos(left) or Jurkat (right), using Btk (D3H5) Rabbit mAb.


Flow Cytometry

Flow Cytometry

Human whole blood was fixed, lysed, and permeabilized as per the Cell Signaling Technology Flow Alternate Protocol and stained using Btk (D3H5) Rabbit mAb. Samples were co-stained using CD3-PE and CD19-APC to distinguish T and B cell subpopulations, respectively. B (red) and T (blue) cell population gates were applied to a histogram depicting the mean fluorescence intensity of Btk. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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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Companion Products

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

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