ab126429 is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in cells. It can be used for measuring the relative amount of STAT5 (Tyr694) phosphorylation and screening the effects of various treatments, inhibitors (such as siRNA or chemicals), or activators in cultured human cell lines. By determining STAT5 protein phosphorylation in your experimental model system, you can verify pathway activation in your cell lines without spending excess time and effort in preparing cell lysate and performing an analysis of Western Blot.
In the In-Cell STAT5 (Tyr694) ELISA kit, cells are seeded into a 96 well tissue culture plate. The cells are fixed after various treatments, inhibitors or activators. After blocking, Anti Phospho-STAT5 (Tyr694) or Anti-STAT5 (primary antibody) is pipetted into the wells and incubated. The wells are washed, and HRP-conjugated anti-mouse IgG (secondary antibody) is added to the wells. The wells are washed again, a TMB substrate solution is added to the wells and color develops in proportion to the amount of protein. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
|Components||1 x 96 tests|
|Anti-Mouse IgG Concentrate (Item I)||1 x 10µl|
|5X Blocking Buffer Concentrate (Item F)||1 x 20ml|
|Fixing Solution (Item D)||1 x 30ml|
|Uncoated 96-well Microplate (Item A)||1 unit|
|Mouse Anti-Phospho-STAT5 (Tyr694) Concentrate (Item G)||1 x 7µl|
|Mouse Anti-STAT5 Concentrate (Item H)||1 x 10µl|
|Quenching Buffer Concentrate (30x) (Item E)||1 x 2ml|
|Stop Solution||1 x 14ml|
|TMB Substrate Reagent||1 x 12ml|
|20X Wash Buffer A Concentrate (Item B)||1 x 30ml|
|20X Wash Buffer B Concentrate (Item C)||1 x 30ml|
Our Abpromise guarantee covers the use of ab126429 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|In-Cell ELISA||Use at an assay dependent concentration.|
A431 cells were stimulated by different concentrations of EGF for 10 minutes at 37°C.
A431 cells were stimulated by different concentrations of EGF for 30 minutes at 37°C.
ab126429 has not yet been referenced specifically in any publications.