Figure 1: Treatment of A-431 cells with EGF stimulates phosphorylation of Stat3 at Ser727, as detected by PathScan® Phospho-Stat3 (Ser727) Sandwich ELISA Kit #7995, but does not affect levels of total Stat3 protein detected by PathScan® Total Stat3 Sandwich ELISA Kit #7305. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Stat3 Antibody #9132 (left panel) or Phospho-Stat3 (Ser727) #9134 (right panel) is shown in the bottom figure.
Figure 2: The relationship between protein concentration of lysates from untreated and EGF-treated A-431 cells and the absorbance at 450 nm is shown. After starvation, A-431 cells (85% confluence) were treated with EGF (100 ng/ml) for 5 min at 37ºC and then lysed.
|REACTIVITY||H M R Mk|
|Product Includes||Volume||Solution Color|
|Stat3 Rabbit mAb Coated Microwells||96 tests|
|Stat3 Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 ea|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Total Stat3 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Stat3 protein. A Stat3 rabbit mAb has been coated onto the microwells. After incubation with cell lysates, Stat3 protein is captured by the coated antibody. Following extensive washing, a Stat3 mouse mAb is added to detect captured Stat3 protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Stat3 protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Total Stat3 Sandwich ELISA Kit detects endogenous levels of Stat3 protein in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Human, Mouse, Rat, Monkey
The Stat3 transcription factor is an important signaling molecule for many cytokines and growth factor receptors (1) and is required for murine fetal development (2). Research studies have shown that Stat3 is constitutively activated in a number of human tumors (3,4) and possesses oncogenic potential (5) and anti-apoptotic activities (3). Stat3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding (6,7). Transcriptional activation seems to be regulated by phosphorylation at Ser727 through the MAPK or mTOR pathways (8,9). Stat3 isoform expression appears to reflect biological function as the relative expression levels of Stat3α (86 kDa) and Stat3β (79 kDa) depend on cell type, ligand exposure, or cell maturation stage (10). It is notable that Stat3β lacks the serine phosphorylation site within the carboxy-terminal transcriptional activation domain (8).
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