Cell Signaling Technology

Product Pathways - Lymphocyte Signaling

Phospho-Syk Antibody Sampler Kit #9925

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-Syk (Tyr323) Antibody #2715 40 µl W IP H (M) 72 Rabbit
Phospho-Syk (Tyr525/526) Antibody #2711 40 µl W H (M) (R) 72 Rabbit
Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody #2701 40 µl W IP IF-IC F H (M) (R) (Hm) (Mk) (C) (B) (Dg) (Pg) (Hr) 70 Zap-70, 72 Syk Rabbit
Syk (D1I5Q) Rabbit mAb #12358 40 µl W IP H M R 72 Rabbit
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

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

Specificity / Sensitivity

Phospho-Syk (Ser323) and Phospho-Syk (Tyr525/526) Antibodies detect tTyr323 and Tyr 525/526 phosphorylated human Syk. Syk Antibody and Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibodies detect endogenous levels of total human Syk and Tyr352 phosphorylated Syk, respectively. Each phospho-Syk antibody recognizes only the specific phosphorylated form of Syk. Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody cross-reacts with the phosphorylated form of Zap-70. All other antibodies in the kit do not cross-react with phosphorylated or nonphosphorylated forms of other related family members.

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 Jurkat cells, starved for 16 hours, and treated with H2O2 (2 mM) or with calf intestinal alkaline phosphatase (CIP), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody #2701 (upper) or control Zap-70 Antibody #2702 (lower).

Western Blotting

Western Blotting

Western blot analysis of cell lysates from 293T cells expressing recombinant Wild-type and mutant Syk proteins (which were activated by cotransfected CD8), using Phospho-Syk (Tyr525/526) Antibody #2711 (upper) and Syk Antibody #2712 (lower). (Provided by Dr. Hamid Band, Harvard University, Massachusetts.)


Western Blotting

Western Blotting

Dephosphorylation of phosphorylated Wild-type Syk abolished Phospho-Syk (Tyr525/526) Antibody's recognition of this protein.

Western Blotting

Western Blotting

Western blot analysis of cell lysates from 293T cells expressing recombinant Wild-type and mutant Syk proteins (which were activated by cotransfected CD8), using Phospho-Syk (Tyr323) Antibody #2715 (upper) and Syk Antibody #2712 (lower). (Provided by Dr. Alagarsamy L. Reddi, laboratory of Dr. Hamid Band, Harvard University, Massachusetts.)

Description

Phospho-Syk Sampler Kit provides an economical means to evaluate the activation status of Syk, including the phosphorylation of Tyr323, Tyr352 and Tyr525/526. The control Syk Antibody is also included. The kit contains enough primary and secondary antibodies for four Western blot experiments.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Tyr323, Tyr352 or Tyr525/526 of human Syk. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Syk (D1I5Q) Rabbit mAb #12358 is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human Syk 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 of a role for Syk in nonimmune cells, and investigators have indicated that Syk is a potential tumor suppressor in human breast carcinomas (5). Tyr323 is a negative regulatory phosphorylation site within the SH2-kinase linker region in Syk. Phosphorylation at Tyr323 provides a direct binding site for the TKB domain of Cbl (6,7). Tyr352 of Syk is involved in the association of PLCγ1 (8). Tyr525 and Tyr526 are located in the activation loop of the Syk kinase domain; phosphorylation at Tyr525/526 of human Syk (equivalent to Tyr519/520 of mouse Syk) is essential for Syk function (9).

  1. Cheng, A.M. and Chan, A.C. (1997) Curr. Opin. Immunol. 9, 528-533.
  2. Kurosaki, T. et al. (1997) Curr. Opin. Immunol. 9, 309-318.
  3. Chu, D.H. et al. (1998) Immunol. Rev. 165, 167-180.
  4. Turner, M. et al. (2000) Immunol. Today 21, 148-154.
  5. Coopman, P.J. et al. (2000) Nature 406, 742-747.
  6. Deckert, M. et al. (1998) J. Biol. Chem. 273, 8867-8874.
  7. Rao, N. et al. (2001) EMBO J. 20, 7085-7095.
  8. Law, C.L. et al. (1996) Mol. Cell. Biol. 16, 1305-1315.
  9. Zhang, J. et al. (2000) J. Biol. Chem. 275, 35442-35447.

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