Microplate assays to measure total and phospho-STAT3 in whole cells.
Thermo Scientific Pierce STAT3 In-Cell ELISA Kits are colorimetric and fluorescent microplate assays optimized to measure total and phosphorylated signal transducers and activators of transcription 3 (phospho-STAT3) in whole cells.
These ELISA kits for whole cells include a mouse monoclonal anti-STAT3 antibody, rabbit monoclonal anti-phospho-(Tyr705) STAT3 antibody, detection reagents and all buffers required for either colorimetric or near infrared detection. The STAT3 In-Cell ELISA Kits enable simultaneous detection of both total and phospho-(Tyr705) STAT3.
Measures total and phosphorylated signal transducers and activators of transcription 3 (STAT3) in whole cells using a 96- or 384-well microplates
colorimetric detection kit for use with standard microplate readers
near infrared fluorescent detection kit for use with IR imaging system
Whole-cell detection requires no cell lysis, protein extraction or special sample preparation
Provides better data and higher throughput than quantitative Western blotting
Less costly (one well vs. one lane) than Western blotting
Stimulation with EGF causes increased STAT3 phosphorylation A431 cells. Relative total and phospho-STAT3 levels were measured in A431 cells using the STAT3 Colorimetric and Near IR In-Cell ELISA Kits. Cells were serum-starved overnight and stimulated with 100ng/ml EFG for five minutes immediately prior to fixation.The level of STAT3 phosphorylation increased 2 fold in response to EGF stimulation as determined by the Near IR In-Cell ELISA Kit (3.3 fold in the colorimetric experiment). Total STAT3 was not significantly different between experiments or treatments. Note: Absolute absorbance values cannot be directly compared between phospho and total STAT3 experiments because they use antibodies with different sensitivities.
Treatment with EGF increases phosphorylation of STAT3 in A431 cells and is blocked in a dose-depended manner with EGFR inhibitor. Relative total and STAT3 levels were measured in A431 cells using the (A) STAT3 Near Infrared In-Cell ELISA Kit. Cells were serum-starved overnight and then treated with EGF for five minutes immediately prior to fixation. The EGF-induced phosphorylation of STAT3 is reduced in a dose-dependent manner when cell receive a two hour pre-treatment with the minute EGFR inhibitor, PD168393 (B, STAT3 Colorimetric In-Cell ELISA Kit). Note: Absolute absorbance values cannot be directly compared between phospho and total STAT3 experiments because they use antibodies with different sensitivities.
Overview of STAT3 and phosphorylation of STAT3:
STAT3 belongs to the STAT family of transcription factors which regulate many aspects of cell growth, survival and differentiation. Inactive STAT proteins are located in the cytosol until phosphorylated by receptor associated kinases. Upon phosphorylation, STAT proteins form homodimers or heterodimers with other STAT proteins and translocate into the cell nucleus. STAT3 is activated in response to cytokines and growth factors such as interons, epidermal growth factors, interleukins and bone morphogenetic protein 2. STAT3 is known to be regulated by Rac1, PIAS3, JAK2 and c-MET. Activation of STAT3 is required for embyronic stem cell survial and development of mouse embryos past embyronic day 7. STAT3 is also implicated in human cancers. The STAT3 In-Cell ELISA Kits enable simultaneous detection of both total and phospho-(Tyr705) STAT3.