Figure 1. Treatment of Ramos cells with goat anti-human IgM stimulates tyrosine phosphorylation of Syk, detected by PathScan® Phospho-Syk (panTyr) Sandwich ELISA Kit #7928, but does not affect the level of total Syk protein detected by western analysis. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots, using Syk Antibody #2712 (left panel), or Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb #2710 (right panel) are shown in the bottom figure.
Figure 2. The relationship between protein concentration of untreated or goat anti-human IgM treated Ramos cell lysates and the absorbance at 450 nm is shown. Cells were serum starved overnight and then treated with goat anti-human IgM (10 μg/ml) for 10 min. at 37ºC.
|Product Includes||Volume||Solution Color|
|Syk Rabbit mAb Coated Microwells||96 tests|
|Phospho Tyrosine Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent 2||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Phospho-Syk (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Syk when phosphorylated at tyrosine residues. A Syk mouse mAb has been coated onto the microwells. After incubation with cell lysates, Syk (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a biotinylated phospho-tyrosine mouse detection antibody is added to detect tyrosine phosphorylation of the captured Syk protein. HRP-linked strepavidin is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Syk phosphorylated on tyrosine.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Phospho-Syk (panTyr) Sandwich ELISA Kit #7928 detects Syk when tyrosine phosphorylated. As shown in Figure 1, a significant induction of Syk tyrosine phosphorylation can be detected in Ramos cells following treatment with anti-human IgM using the Phospho-Syk (panTyr) Sandwich ELISA Kit #7928. The level of total Syk (phospho and nonphospho) remains unchanged as shown by western analysis. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
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).