Description

STAT1 (Phospho-Ser727) Cell-Based ELISA Kit

The STAT1 (Phospho-Ser727) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor STAT1 phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated STAT1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on STAT1 phosphorylation.

How does our STAT1 (Phospho-Ser727) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of STAT1 (Phospho-Ser727) concentration is achieved by an indirect ELISA format. In essence, STAT1 (Phospho-Ser727) is captured by STAT1 (Phospho-Ser727)-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 STAT1 (Phospho-Ser727) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated STAT1 can be used to normalize the RFU value for phosphorylated STAT1.

STAT1 (Phospho-Ser727) Fluorometric Cell-Based ELISA Kit -Information

Product Name:	STAT1 (Phospho-Ser727) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:	FBCAB00079
Description:	The STAT1 (Phospho-Ser727) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor STAT1 (Phospho-Ser727) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated STAT1 (Phospho-Ser727) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on STAT1 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: 6772, UniProt ID: P42224, OMIM #: 209950/600555, Unigene #: Hs.642990
Format:	Two 96-Well Plates
NCBI Gene Symbol:	STAT1
Sub Type:	Phospho
Target Name:	Phospho-STAT1 (Ser727)

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 STAT1 (Phospho-Ser727) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total STAT1 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.

STAT1 (Phospho-Ser727) - Protein Information

UniProt Protein Function:	STAT1: transcription factor of the STAT family. Phosphorylated and activated by receptor-associated kinases downstream of certain receptor tyrosine kinases, GPCRs, and receptors for various interleukins and interferons. Forms homo- or heterodimers that translocate into the nucleus where they regulate transcription. Two alternatively spliced isoforms have been described.
UniProt Protein Details:	Protein type:DNA-binding; Transcription factor Chromosomal Location of Human Ortholog: 2q32.2 Cellular Component: axon; cytoplasm; cytosol; dendrite; nuclear chromatin; nucleolus; nucleoplasm; nucleus; perinuclear region of cytoplasm Molecular Function:double-stranded DNA binding; enzyme binding; identical protein binding; protein binding; protein homodimerization activity; signal transducer activity; transcription factor activity; tumor necrosis factor receptor binding Biological Process: apoptosis; blood circulation; defense response to virus; endothelial cell migration; JAK-STAT cascade; negative regulation of angiogenesis; negative regulation of endothelial cell proliferation; negative regulation of I-kappaB kinase/NF-kappaB cascade; negative regulation of transcription from RNA polymerase II promoter; negative regulation of viral protein levels in host cell; positive regulation of mesenchymal cell proliferation; positive regulation of smooth muscle cell proliferation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; regulation of apoptosis; regulation of transcription from RNA polymerase II promoter; response to cAMP; response to cytokine stimulus; response to peptide hormone stimulus; transcription, DNA-dependent; tumor necrosis factor-mediated signaling pathway; viral reproduction Disease: Immunodeficiency 31a; Immunodeficiency 31b; Immunodeficiency 31c
NCBI Summary:	The protein encoded by this gene is a member of the STAT protein family. 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 can be activated by various ligands including interferon-alpha, interferon-gamma, EGF, PDGF and IL6. This protein mediates the expression of a variety of genes, which is thought to be important for cell viability in response to different cell stimuli and pathogens. Two alternatively spliced transcript variants encoding distinct isoforms have been described. [provided by RefSeq, Jul 2008]
UniProt Code:	P42224
NCBI GenInfo Identifier:	2507413
NCBI Gene ID:	6772
NCBI Accession:	P42224.2
UniProt Secondary Accession:	P42224,Q53S88, Q53XW4, Q68D00, Q9UDL5, A8K989, B2RCA0 D2KFR8, D3DPI7,
UniProt Related Accession:	P42224
Molecular Weight:
NCBI Full Name:	Signal transducer and activator of transcription 1-alpha/beta
NCBI Synonym Full Names:	signal transducer and activator of transcription 1
NCBI Official Symbol:	STAT1
NCBI Official Synonym Symbols:	CANDF7; IMD31A; IMD31B; IMD31C; ISGF-3; STAT91
NCBI Protein Information:	signal transducer and activator of transcription 1-alpha/beta
UniProt Protein Name:	Signal transducer and activator of transcription 1-alpha/beta
UniProt Synonym Protein Names:	Transcription factor ISGF-3 components p91/p84
Protein Family:	Signal transducer and activator of transcription
UniProt Gene Name:	STAT1
UniProt Entry Name:	STAT1_HUMAN