Immunofluorescence analysis of Phospho-Syk pTyr323 / pTyr317 was performed using 70% confluent log phase Jurkat cells treated with 100 uM H2O2 for 1 hour. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Phospho-Syk pTyr323 / pTyr317 Rabbit Polyclonal Antibody (44234G) at 2 ug/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows untreated cells with no signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
|Tested species reactivity||Human, Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human Syk that contains tyrosine 323 (tyrosine 317 in the mouse sequence).|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 50% glycerol, 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Immunocytochemistry (ICC)||2-3 µg/ml|
|Immunofluorescence (IF)||2-3 µg/ml|
|Western Blot (WB)||Assay Dependent|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
SYK is a positive effector of BCR stimulated responses. It couples the B cell antigen receptor (BCR) to the mobilization of calcium ions either through a phosphoinositide 3 kinase dependent pathway, when not phosphorylated on tyrosines of the linker region, or through a phospholipase C gamma dependent pathway, when phosphorylated on Tyr 342 and Tyr 346. Thus the differential phosphorylation of SYK can determine the pathway by which BCR is coupled to the regulation of intracellular calcium ions.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.