Cell Signaling Technology

Product Pathways - Lymphocyte Signaling

B Cell Signaling Antibody Sampler Kit #9768

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb #2710 40 µl W IP F H (M) (R) 72 kDa Rabbit IgG
Syk (D1I5Q) Rabbit mAb #12358 40 µl W IP H M R 72 Rabbit
Phospho-Lyn (Tyr507) Antibody #2731 40 µl W H M (R) 53, 56 Rabbit
Lyn Antibody #2732 40 µl W IP H M R 56 Rabbit
Phospho-Btk (Tyr223) Antibody #5082 40 µl W H (M) (R) 77 Rabbit
Btk (D3H5) Rabbit mAb #8547 40 µl W IP IHC-P F H M (R) (Hm) (Mk) (B) (Dg) (Pg) (Hr) 77 Rabbit IgG
Phospho-CD19 (Tyr531) Antibody #3571 40 µl W IP H 95 Rabbit
CD19 Antibody #3574 40 µl W IP IF-IC F H M 95 Rabbit
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

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

Specificity / Sensitivity

Syk Antibody detects endogenous levels of total Syk protein near the amino terminus of human Syk protein. Syk (D1I5Q) Rabbit mAb recognizes endogenous levels of total Syk protein. Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb detects endogenous levels of Syk protein only when phosphorylated at Tyr525/526 of human Syk or Tyr519/520 of mouse Syk. It also detects Syk protein when singly phosphorylated at Tyr526 of human Syk or Tyr520 of mouse Syk, but does not cross-react with other tyrosine-phosphorylated protein tyrosine kinases. Lyn Antibody detects endogenous levels of the 56 kDa isoform of Lyn, but does not recognize the 53 kDa Lyn isoform. Phospho-Lyn (Tyr507) Antibody detects endogenous levels of Lyn only when phosphorylated at Tyr507. This antibody may cross-react with phospho-Lck (Tyr505) and phospho-Src (Tyr527) due to high sequence homology. Btk (D3H5) Rabbit mAb recognizes endogenous levels of total Btk protein. Phospho-Btk (Tyr223) Antibody detects endogenous levels of Btk only when phosphorylated at Tyr223. CD19 Antibody detects endogenous levels of total CD19 protein. Phospho-CD19 (Tyr531) Antibody detects endogenous levels of CD19 only when phosphorylated at Tyr531.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Syk (D1I5Q) Rabbit mAb #12358.

Western Blotting

Western Blotting

Western blot analysis of extracts from Ramos cells, untreated or treated with anti-IgM, using Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb #2710 (upper), or Syk Antibody #2712 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from Ramos cells, treated with anti-IgM antibody (12 µg/ml for 2 minutes), using Phospho-Lyn (Tyr507) Antibody #2731. The phospho-specificity of the antibody was characterized by treating the membrane without or with calf intestinal alkaline phosphatase (CIP) after western transfer.


Western Blotting

Western Blotting

Western blot analysis of extracts from Ramos cells, untreated or anti-human IgM-treated (12 µg/ml for 2 minutes), using Phospho-CD19 (Tyr531) Antibody #3571 (upper) and CD19 Antibody #3574 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from Ramos cells, untreated or anti-human IgM-treated (12 µg/ml for 2 minutes), and A20 cells, untreated or anti-mouse IgG-treated (30 µg/ml for 2 minutes), using CD19 Antibody #3574.

Western Blotting

Western Blotting

Western blot analysis of extracts from Ramos cells, untreated or anti-human IgM-treated (12 µg/ml for 10 minutes), using Phospho-Btk (Tyr223) Antibody #5082 (upper) or Btk (C82B8) Rabbit mAb #3533 (lower).


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Btk (D3H5) Rabbit mAb #8547.

Western Blotting

Western Blotting

Western blot analysis of extracts from Ramos cells, untreated or treated with anti-IgM (12 µg/ml for 2 minutes), using Lyn Antibody #2732.

Description

The B Cell Signaling Antibody Sampler Kit provides an economical means to examine key signaling proteins commonly associated with B cell activation. The provided antibodies allow monitoring of both total protein levels and the phosphorylation state of each target. The kit includes enough primary and secondary antibody to perform four western mini-blot experiments.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a nonphosphorylated synthetic peptide corresponding to residues around the carboxy-terminus of human CD19, and the amino-terminal sequence of human Lyn. Phospho-specific polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr507 of human Lyn, Tyr223 of human Btk, and Tyr531 of human CD19. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Rabbit monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues near the amino terminus of human Syk, surrounding Tyr525/526 of human Syk and to residues surrounding Asp195 of human Btk protein.

Background

Syk is a protein tyrosine kinase that plays an important role in intracellular signal transduction in hematopoietic cells (1-3). Syk interacts with immunoreceptor tyrosine-based activation motifs (ITAMs) located in the cytoplasmic domains of immune receptors (4). It couples the activated immunoreceptors to downstream signaling events that mediate diverse cellular responses, including proliferation, differentiation, and phagocytosis (4). There is also evidence that Syk plays a role in nonimmune cells; Syk is a potential tumor suppressor in human breast carcinomas (5). Tyrosine 525 and 526 are located in the activation loop of the Syk kinase domain, and phosphorylation of Tyr525/526 of human Syk (equivalent to the Tyr519/520 of mouse Syk) is essential for Syk function (6).Lyn, one of the Src family members, is predominantly expressed in hematopoietic cells (7). Two tyrosine residues have been reported to play a crucial role in the regulation of protein tyrosine kinases of the Src family. Autophosphorylation of Tyr396 (equivalent to Tyr416 of Src), located in the catalytic domain, correlates with enzyme activation. Csk-mediated phosphorylation of the carboxy-terminal Tyr507 (equivalent to Tyr527 of Src) inactivates the kinase. Tyrosine phosphorylation and activation of Lyn occurs upon association with cell surface receptors such as the B cell Ag receptor (BCR) and CD40 (8-10).Bruton's tyrosine kinase (Btk) is a member of the Btk/Tec family of cytoplasmic tyrosine kinases. Btk plays an important role in B cell development (11,12). 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 (13-15). 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 (16,17).CD19 is a 95 kDa coreceptor that amplifies the signaling cascade in B cells (18). On the B cell surface, CD19 associates with CD21, CD81, and Leu-13 to exert its function. The cytoplasmic tail of CD19 has nine conserved tyrosine residues playing critical roles in CD19-mediated function by coupling signaling molecules to the receptor (18). After BCR or CD19 ligation, Tyr531 and Tyr500 of CD19 are progressively phosphorylated. This phosphorylation enables the coupling of PI3 kinase and Src family tyrosine kinase to CD19 and activates the PI3K and Src signaling pathways (19,20).

  1. Cheng, A.M. and Chan, A.C. (1997) Curr Opin Immunol 9, 528-33.
  2. Kurosaki, T. (1997) Curr Opin Immunol 9, 309-18.
  3. Chu, D.H. et al. (1998) Immunol Rev 165, 167-80.
  4. Turner, M. et al. (2000) Immunol Today 21, 148-54.
  5. Coopman, P.J. et al. (2000) Nature 406, 742-7.
  6. Zhang, J. et al. (2000) J Biol Chem 275, 35442-7.
  7. Yamanashi, Y. et al. (1989) Proc Natl Acad Sci U S A 86, 6538-42.
  8. Yamanashi, Y. et al. (1991) Science 251, 192-4.
  9. Burkhardt, A.L. et al. (1991) Proc Natl Acad Sci U S A 88, 7410-4.
  10. Ren, C.L. et al. (1994) J Exp Med 179, 673-80.
  11. Khan, W.N. (2001) Immunol Res 23, 147-56.
  12. Lewis, C.M. et al. (2001) Curr Opin Immunol 13, 317-25.
  13. Salim, K. et al. (1996) EMBO J 15, 6241-50.
  14. Rameh, L.E. et al. (1997) J Biol Chem 272, 22059-66.
  15. Várnai, P. et al. (1999) J Biol Chem 274, 10983-9.
  16. Rawlings, D.J. et al. (1996) Science 271, 822-5.
  17. Park, H. et al. (1996) Immunity 4, 515-25.
  18. Tedder, T.F. et al. (1997) Immunity 6, 107-18.
  19. Buhl, A.M. and Cambier, J.C. (1999) J Immunol 162, 4438-46.
  20. Fujimoto, M. et al. (2000) Immunity 13, 47-57.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Protocols

* Product-specific protocol.

Companion Products

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

Products