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Direct ELISA analysis of Phosphotyrosine, Phosphoserine, and Phosphothreonine was performed by coating wells of a 96-well plate with 100ul per well of Phosphotyrosine, Phosphoserine or Phosphothreonine-BSA conjugate diluted in carbonate/bicarbonate buffer (Product # 28382) at a concentration of 5ug/ml overnight at 4°C. Wells of the plate were washed, blocked with Superblock blocking buffer (Product # 37515), and incubated with 100ul per well of a rabbit anti-phosphotyrosine,serine and threonine polyclonal antibody (Product # 61-8300) at a starting concentration of 1ug/ml and serially diluting 2-fold to a concentration of 8ng/ml for 1 hour at 37C. The plate was washed, then incubated with 100ul per well of an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31430) at a dilution of 1:8000 for 30 minutes at 37C. Detection was performed using 1-Step Ultra TMB substrate (Product # 34028) for 5 minutes at room temperature in the dark. The reaction was stopped with 0.16M sulfuric acid, and absorbances were read on a spectrophotometer at 450-550nm.
|Tested species reactivity||Chem|
|Published species reactivity||Primate, Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Phosphothreonine conjugated to protein carriers|
|Storage buffer||PBS, pH 7.4|
|Contains||0.1% sodium azide|
|Tested Applications||Dilution *|
|ELISA (ELISA)||1 µg/ml|
|Immunoprecipitation (IP)||5-10 ug|
|Western Blot (WB)||1.0 ug/ml|
* 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.
|Western Blot (WB)||See 2 publications below|
This antibody detects pS, pT and pY amino acids with very low cross reactivity to their non-phosphorylated variants
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Primate||Not Cited||Conserved SNH domain of the proto-oncoprotein SYT interacts with components of the human chromatin remodelling complexes, while the QPGY repeat domain forms homo-oligomers.||Perani M,Ingram CJ,Cooper CS,Garrett MD,Goodwin GH||Oncogene (22:8156)||2003|
|Mouse||Not Cited||IkappaB kinase-independent IkappaBalpha degradation pathway: functional NF-kappaB activity and implications for cancer therapy.||Tergaonkar V,Bottero V,Ikawa M,Li Q,Verma IM||Molecular and cellular biology (23:8070)||2003|
phosphoserine; phosphothreonine; phosphotyrosine; pSer; pThr; pTyr; serine; threonine; tyrosine