STAT3 [pY705] Phospho-ELISA Kit, Multispecies

Catalog number: KHO0481

Novex™  Related applications: Protein Assays and Analysis

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Description

The STAT3 [pY705] ELISA research-use-only kit is designed to detect and quantify the level of STAT3 protein phosphorylated at tyrosine residue 705. This assay is intended for the detection of human, mouse, and rat STAT3 [pY705] from cell or tissue homogenates using 96-well plates and a microplate reader.

Performance characteristics
• Sensitivity: <0.9 Units/mL
• Standard curve range: 1.57–100 Units/mL
• Sample types: cell lysates, tissue homogenates
• Species cross-reactivity: human, mouse, rat
• Sample volume: 100 μL (1:10)
• Total assay incubation time: 4 hrs

Principle of the method
The STAT3 [pY705] kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA). A monoclonal antibody specific for STAT3 has been coated onto the wells of the microtiter strips provided. Samples, including a standard containing STAT3 [pY705], control specimens, and unknowns, are pipetted into these wells. During the first incubation, the STAT3 antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for STAT3 phosphorylated at tyrosine 705 is added to the wells.

During the second incubation, this antibody serves as a detection antibody by binding to the immobilized STAT3 protein captured during the first incubation. After removal of excess detection antibody, a horseradish peroxidase-labeled anti-rabbit IgG (anti-rabbit IgG-HRP) is added. This binds to the detection antibody to complete the four-member sandwich. After a third incubation and washing to remove all the excess anti-rabbit IgG-HRP, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of STAT3 [pY705] present in the original specimen.

Background information
STAT3 (also known as acute phase response factor) is a member of the STAT family of proteins, which is comprised of STATs 1, 2, 3, 4, 5A, 5B, and 6. Two STAT3 isoforms have been identified, designated STAT3α (full length form) and STAT3β (a truncated form in which the C-terminal 55 amino acid residues have been replaced by an unrelated 5 amino acid–residue fragment). STAT proteins control the transcription of specific genes by binding to a DNA consensus sequence (TTN5AA). Genes under the control of STAT3 include those for acute phase proteins (C-reactive protein, tissue inhibitor of metalloproteinases, and others), JunB, IRF-1, cyclin D1, c-Myc, Bcl-xL, Mcl-1, VEGF, survivin, as well as STAT3 itself.

STAT3 activation occurs via tyrosine phosphorylation at Tyr 705. Tyrosine phosphorylation in response to cytokine stimulation is mediated by a Janus kinase, most often JAK1. Upon activation, STAT3 dimerizes, translocates to the nucleus, and binds DNA response elements thereby influencing gene expression profiles. In addition to tyrosine phosphorylation, STAT3 is also phosphorylated at Ser 727 by the mitogen-activated protein kinases (MAPKs) ERK1&2, p38 MAPK, and JNK, and possibly by other, as yet undefined kinases, as evidenced by sensitivity to the protein kinase inhibitor H7. Phosphorylation at Ser 727 enhances STAT3’s transcriptional activation by promoting recruitment and interaction with transcription cofactors. STATs can also be activated by growth factor receptors, particularly the EGFR, as well as by members of the Src family of kinases.

Detect low-expressing proteins and use less sample
ELISA kits designed to measure intracellular signaling targets are typically 2–10 times more sensitive than western blotting. The improved sensitivity enables you to detect low-expressing proteins that otherwise may not be distinguishable from background. In addition, the amount of sample needed to run the assay is less than what is needed for western blots.

Each STAT3 [pY705] ELISA kit is validated for sensitivity, specificity, precision, and lot-to-lot consistency. See product insert for more information on validation.

Related links:
Learn more about ELISA kits
Learn more about other immunoassays
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Gene Aliases: APRF, FLJ20882, HIES, MGC16063
Accession Number: NP_998827.1, NP_644805.1, NM_213662.1, NM_139276.2, NM_003150.3, NP_003141.2
Shipping Condition: Wet Ice
Marker: STAT3
Gene ID: 6774
Species: Mouse, Rat, Human
Gene Symbol: STAT3
Sensitivity: <0.9 U⁄mL
Target Gene: signal transducer and activator of transcription 3 (acute-phase response factor)
Label or Dye: HRP (Horseradish Peroxidase)
Product Size: 96 assays
Concentration: 1.6 U⁄ml
Sample Volume: 10 µl
Protein Family: Transcription Factors
Incubation Time: 4 hrs
Detection Method: Colorimetric
Research Category: Signal Transduction
Sample Type (Specific): Cell Extracts
For Use With (Equipment): Microplate Reader

Contents & storage

Pre-coated plate(s), standard or calibrator, detector antibody, HRP conjugate, diluents, wash buffer, substrate(s), and stop solution. Store kit at 2-8°C. See product manual for detailed contents and storage conditions for maximum stability.

Documents

Manuals & protocols