FOXO1A (Phospho-Ser256) Fluorometric Cell-Based ELISA Kit

SKU:

FBCAB00066

Product Type:

ELISA Kit

ELISA Type:

Cell Based Phospho Specific

Reactivity:

Human

Mouse

Rat

Detection Method:

Fluorometric

Description

Description

FOXO1A (Phospho-Ser256) Cell-Based ELISA Kit

The FOXO1A (Phospho-Ser256) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor FOXO1A phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated FOXO1A in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on FOXO1A phosphorylation.

How does our FOXO1A (Phospho-Ser256) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of FOXO1A (Phospho-Ser256) concentration is achieved by an indirect ELISA format. In essence, FOXO1A (Phospho-Ser256) is captured by FOXO1A (Phospho-Ser256)-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 FOXO1A (Phospho-Ser256) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated FOXO1A can be used to normalize the RFU value for phosphorylated FOXO1A.

FOXO1A (Phospho-Ser256) Fluorometric Cell-Based ELISA Kit -Information

Product Name:	FOXO1A (Phospho-Ser256) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:	FBCAB00066
Description:	The FOXO1A (Phospho-Ser256) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor FOXO1A (Phospho-Ser256) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated FOXO1A (Phospho-Ser256) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on FOXO1 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: 2308, UniProt ID: Q12778, OMIM #: 136533/268220, Unigene #: Hs.370666
Format:	Two 96-Well Plates
NCBI Gene Symbol:	FOXO1
Sub Type:	Phospho
Target Name:	Phospho-FOXO1A (Ser256)

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 FOXO1A (Phospho-Ser256) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total FOXO1A 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.

FOXO1A (Phospho-Ser256) - Protein Information

UniProt Protein Function:	FOXO1A: a transcription factor of the forkhead family. A central regulator of metabolism in several cell types. May play an important role in myogenic growth and differentiation, and in the regulation of lipolysis in adipocytes by controlling the expression of adipose triglyceride lipase (ATGL), the rate-limiting lipolytic enzyme. Translocation of this gene with PAX3 has been associated with alveolar rhabdomyosarcoma. Contains 1 fork-head domain.
UniProt Protein Details:	Protein type:Nuclear receptor co-regulator; Oncoprotein; DNA-binding; Transcription factor Chromosomal Location of Human Ortholog: 13q14.1 Cellular Component: cytoplasm; cytosol; mitochondrion; nucleoplasm; nucleus Molecular Function:beta-catenin binding; chromatin binding; protein binding; protein phosphatase 2A binding; sequence-specific DNA binding; ubiquitin protein ligase binding Biological Process: anatomical structure morphogenesis; apoptosis; autophagy; blood vessel development; cell differentiation; cell glucose homeostasis; cellular response to insulin stimulus; cellular response to starvation; endocrine pancreas development; epidermal growth factor receptor signaling pathway; fat cell differentiation; fibroblast growth factor receptor signaling pathway; innate immune response; insulin receptor signaling pathway; negative regulation of apoptosis; negative regulation of fat cell differentiation; negative regulation of stress-activated MAPK cascade; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; nerve growth factor receptor signaling pathway; phosphoinositide-mediated signaling; positive regulation of apoptosis; positive regulation of autophagy; positive regulation of gluconeogenesis; positive regulation of protein catabolic process; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; protein amino acid acetylation; response to DNA damage stimulus; thermoregulation; transcription, DNA-dependent Disease: Rhabdomyosarcoma 2
NCBI Summary:	This gene belongs to the forkhead family of transcription factors which are characterized by a distinct forkhead domain. The specific function of this gene has not yet been determined; however, it may play a role in myogenic growth and differentiation. Translocation of this gene with PAX3 has been associated with alveolar rhabdomyosarcoma. [provided by RefSeq, Jul 2008]
UniProt Code:	Q12778
NCBI GenInfo Identifier:	116241368
NCBI Gene ID:	2308
NCBI Accession:	Q12778.2
UniProt Secondary Accession:	Q12778,O43523, Q5VYC7, Q6NSK6,
UniProt Related Accession:	Q12778
Molecular Weight:
NCBI Full Name:	Forkhead box protein O1
NCBI Synonym Full Names:	forkhead box O1
NCBI Official Symbol:	FOXO1
NCBI Official Synonym Symbols:	FKH1; FKHR; FOXO1A
NCBI Protein Information:	forkhead box protein O1
UniProt Protein Name:	Forkhead box protein O1
UniProt Synonym Protein Names:	Forkhead box protein O1A; Forkhead in rhabdomyosarcoma
Protein Family:	Foxo1-corepressor
UniProt Gene Name:	FOXO1
UniProt Entry Name:	FOXO1_HUMAN