STAT4 (Phospho-Tyr693) Fluorometric Cell-Based ELISA Kit

SKU:

FBCAB00008

Product Type:

ELISA Kit

ELISA Type:

Cell Based Phospho Specific

Reactivity:

Human

Mouse

Rat

Detection Method:

Fluorometric

Description

Description

STAT4 (Phospho-Tyr693) Cell-Based ELISA Kit

The STAT4 (Phospho-Tyr693) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor STAT4 phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated STAT4 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on STAT4 phosphorylation.

How does our STAT4 (Phospho-Tyr693) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of STAT4 (Phospho-Tyr693) concentration is achieved by an indirect ELISA format. In essence, STAT4 (Phospho-Tyr693) is captured by STAT4 (Phospho-Tyr693)-specific primary (1°) antibodies while Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. 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 RFU values.
2.	An antibody against the nonphosphorylated counterpart of STAT4 (Phospho-Tyr693) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated STAT4 can be used to normalize the RFU value for phosphorylated STAT4.

STAT4 (Phospho-Tyr693) Fluorometric Cell-Based ELISA Kit -Information

Product Name:	STAT4 (Phospho-Tyr693) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:	FBCAB00008
Description:	The STAT4 (Phospho-Tyr693) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor STAT4 (Phospho-Tyr693) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated STAT4 (Phospho-Tyr693) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on STAT4 phosphorylation.
Dynamic Range:	> 5000 Cells
Detection Method:	Fluorometric
Storage/Stability:	4°C/6 Months
Reactivity:	Human, Mouse, Rat
Assay Type:	Cell-Based ELISA
Database Links:	Gene ID: 6775, UniProt ID: Q14765, OMIM #: 180300/600558/612253, Unigene #: Hs.80642
Format:	Two 96-Well Plates
NCBI Gene Symbol:	STAT4
Sub Type:	Phospho
Target Name:	Phospho-STAT4 (Tyr693)

Kit Principle

Figure: Schematic representation of Assay Genie Cell-Based Fluorometric ELISA principle

Kit components	Quantity
96-Well Black Cell CultureClear-Bottom Microplate	2 plates
10X TBS	24 ml
Quenching Buffer	24 ml
Blocking Buffer	50 ml
15X Wash Buffer	50 ml
Primary Antibody Diluent	12 ml
100x Anti-Phospho Target Antibody	60 µl
100x Anti-Target Antibody	60 µl
Anti-GAPDH Antibody	110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody	6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody	6 ml
Adhesive Plate Seals	2 seals

Additional equipment and materials required

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

Fluorescent plate reader with two channels at Ex/Em: 651/667 and 495/521
Micropipettes capable of measuring volumes from 1 µl to 1 ml
Deionized or sterile water (ddH2O)
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)

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

1.	Seed 200 µl of desired cell concentration in culture medium into each well of the 96-well plates. 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 3 minutes. 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 3 minutes each time.
9.	Dispense 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 3 minutes each time.
11.	Add 50 µl of Primary Antibody Mixture P to corresponding wells for STAT4 (Phospho-Tyr693) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total STAT4 detection. Cover the plate 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 3 minutes each time.
13.	Add 50 ul of Secondary Antibody Mixture to corresponding wells and incubate for 1.5 hours at room temperature in the dark.
14.	Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
15.	Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.
16.	Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.

STAT4 (Phospho-Tyr693) - Protein Information

UniProt Protein Function:	STAT4: transcription factor of the STAT family. Phosphorylated and activated by receptor-associated kinases downstream of many cytokines and growth-factor receptors. Essential for mediating responses to IL12 in lymphocytes, and regulating the differentiation of T helper cells.
UniProt Protein Details:	Protein type:Transcription factor; DNA-binding Chromosomal Location of Human Ortholog: 2q32.2-q32.3 Cellular Component: nucleoplasm; cytoplasm Molecular Function:protein binding; signal transducer activity; sequence-specific DNA binding; transcription factor activity Biological Process: cell proliferation; transcription, DNA-dependent; cytokine and chemokine mediated signaling pathway; positive regulation of transcription from RNA polymerase II promoter; JAK-STAT cascade; protein amino acid phosphorylation Disease: Systemic Lupus Erythematosus, Susceptibility To, 11
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 is essential for mediating responses to IL12 in lymphocytes, and regulating the differentiation of T helper cells. Mutations in this gene may be associated with systemic lupus erythematosus and rheumatoid arthritis. Alternate splicing results in multiple transcript variants that encode the same protein. [provided by RefSeq, Aug 2011]
UniProt Code:	Q14765
NCBI GenInfo Identifier:	6226158
NCBI Gene ID:	6775
NCBI Accession:	Q14765.1
UniProt Secondary Accession:	Q14765,Q96NZ6,
UniProt Related Accession:	Q14765
Molecular Weight:	85,941 Da
NCBI Full Name:	Signal transducer and activator of transcription 4
NCBI Synonym Full Names:	signal transducer and activator of transcription 4
NCBI Official Symbol:	STAT4
NCBI Official Synonym Symbols:	SLEB11
NCBI Protein Information:	signal transducer and activator of transcription 4
UniProt Protein Name:	Signal transducer and activator of transcription 4
Protein Family:	Protein slender lobes
UniProt Gene Name:	STAT4
UniProt Entry Name:	STAT4_HUMAN