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STAT6 (Phospho-Thr645) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00259
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS
Product Name:STAT6 (Phospho-Thr645) Colorimetric Cell-Based ELISA
Product Code:CBCAB00259
ELISA Type:Cell-Based
Target:STAT6 (Phospho-Thr645)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The STAT6 (Phospho-Thr645) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect STAT6 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated STAT6 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on STAT6 phosphorylation.

Qualitative determination of STAT6 (Phospho-Thr645) concentration is achieved by an indirect ELISA format. In essence, STAT6 (Phospho-Thr645) is captured by STAT6 (Phospho-Thr645)-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 6778, UniProt ID: P42226, OMIM: 601512, Unigene: Hs.524518
Gene Symbol:STAT6
Sub Type:Phospho
UniProt Protein Function:STAT6: transcription factor of the STAT family. Plays a central role in IL4-mediated biological responses. Induces the expression of BCL2L1/BCL-X(L), which is responsible for the anti-apoptotic activity of IL4. May function in the differentiation of T helper 2 cells and class switch of immunoglobulins. Forms homo- or heterodimers that translocate into the nucleus where they regulate transcription.
UniProt Protein Details:

Protein type:DNA-binding; Transcription factor

Chromosomal Location of Human Ortholog: 12q13

Cellular Component: cytoplasm; cytosol; lipid raft; nuclear chromatin; nucleoplasm

Molecular Function:identical protein binding; protein binding; protein phosphatase binding; signal transducer activity; transcription factor activity

Biological Process: mammary gland epithelial cell proliferation; negative regulation of T-helper 2 type immune response; negative regulation of transcription from RNA polymerase II promoter; positive regulation of interferon type I production; positive regulation of isotype switching to IgE isotypes; positive regulation of transcription from RNA polymerase II promoter; regulation of cell proliferation; regulation of transcription from RNA polymerase II promoter; signal transduction; T-helper 1 cell lineage commitment; transcription, DNA-dependent

NCBI Summary:The protein encoded by this gene is a member of the STAT family of transcription factors. In response to cytokines and growth factors, STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators. This protein plays a central role in exerting IL4 mediated biological responses. It is found to induce the expression of BCL2L1/BCL-X(L), which is responsible for the anti-apoptotic activity of IL4. Knockout studies in mice suggested the roles of this gene in differentiation of T helper 2 (Th2) cells, expression of cell surface markers, and class switch of immunoglobulins. Alternative splicing results in multiple transcript variants.[provided by RefSeq, May 2010]
UniProt Code:P42226
NCBI GenInfo Identifier:1174459
NCBI Gene ID:6778
NCBI Accession:P42226.1
UniProt Secondary Accession:P42226,Q5FBW5, Q71UP4, A8K316, B7ZA27, F5GXI9,
UniProt Related Accession:P42226
Molecular Weight:
NCBI Full Name:Signal transducer and activator of transcription 6
NCBI Synonym Full Names:signal transducer and activator of transcription 6
NCBI Official Symbol:STAT6  
NCBI Official Synonym Symbols:STAT6B; STAT6C; D12S1644; IL-4-STAT  
NCBI Protein Information:signal transducer and activator of transcription 6
UniProt Protein Name:Signal transducer and activator of transcription 6
UniProt Synonym Protein Names:IL-4 Stat
Protein Family:Signal transducer and transcription activator
UniProt Gene Name:STAT6  
UniProt Entry Name:STAT6_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µL of 1x primary antibodies Anti-STAT6 (Phospho-Thr645) Antibody, Anti-STAT6 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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