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.
|7929S||1 Kit (Reagents for 4 x 96 well plates)||$ 469|
|Product Includes||Volume||Cap Color|
|Syk Capture Ab (100X)||400 µl||Pink|
|Phospho-Tyrosine Detection Mouse mAb (100X)||400 µl||Blue|
|Anti-mouse IgG, HRP-linked Antibody (1000X)||40 µl||Yellow|
Capture and detection antibodies are stored at 4°C. HRP-linked secondary reagent is stored at -20°C.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).
Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.
STOP Solution: (#7002)
NOTE: Reagents should be made fresh daily.
posted January 2008
revised Sepetember 2013
Protocol Id: 20
CST's PathScan® Phospho-Syk (panTyr) Sandwich ELISA Antibody Pair is being offered as an economical alternative to our PathScan® Phospho-Syk (panTyr) Sandwich ELISA Kit #7928. Capture and detection antibodies (100X stocks) and HRP-linked streptavidin (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The Syk mouse capture antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by biotinylated phospho-tyrosine mouse detection antibody and HRP-linked streptavidin. HRP substrate (TMB) is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of Syk phosphorylated on tyrosines.
For Antibody Pair specificity and sensitivity, please refer to the corresponding PathScan® Sandwich ELISA Kit. Note: This antibody pair 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).