Direct ELISA analysis of Phosphotyrosine was performed by coating wells of a 96-well plate with 100ul per well of Phosphotyrosine or Phosphoserine diluted in carbonate/bicarbonate buffer (Product # 28382) at a concentration of 5ug/ml overnight at 4C. Wells of the plate were washed, blocked with StartingBlock blocking buffer (Product # 37538), and incubated with 100ul per well of a mouse anti-phosphotyrosine monoclonal antibody (Product # 03-7700) at a starting concentration of 1ug/ml and serially diluting 2-fold to a concentration of 31ng/ml for 30 minutes at 37C. The plate was washed, then incubated with 100ul per well of an HRP-conjugated goat anti-mouse 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||Chemical|
|Host / Isotype||Mouse / IgG2b|
|Immunogen||Keyhole limpet hemocyanin coupled with equal amounts of phosphotyrosine, glycine, and alanine|
|Storage buffer||PBS, pH 7.4|
|Contains||0.1% sodium azide|
|Tested Applications||Dilution *|
|ELISA (ELISA)||1 µg/ml|
|Immunofluorescence (IF)||Assay Dependent|
|Immunoprecipitation (IP)||2-5 ug|
|Western Blot (WB)||0.5-1 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.
03-7700 has been successfully used in ELISA, WB, and IP applications, and detects proteins which contain phosphorylated tyrosine residues.
The role of tyrosine phosphorylation in transduction of the mitogenic signal from transmembrane receptors and in transformation by oncogene tyrosine kinases has been the subject of intense investigation for several years. While the phosphorylation of specific tyrosine residues has been shown to be a primary mechanism of signal transduction during normal mitogenesis, cell cycle progression and oncogenic transformation, its role in other areas such as differentiation and gap junction communication, is a matter of active and ongoing research. Antibodies that specifically recognize phosphorylated tyrosine residues have proved to be invaluable to the study of tyrosine -phosphorylated proteins and the biochemical pathways in which they function. The fluorescein (FITC) conjugate of clone PY20 anti-phosphotyrosine is especially useful for the detection of these P-Tyr proteins in immunohistochemical and immunocytochemical protocols in situations wherein the use of a secondary antibody would complicate detection of the protein(s) of interest.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.