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FKHRL1 (Phospho-Ser253) Fluorometric Cell-Based ELISA Kit

SKU:
FBCAB00068
Product type:
ELISA Kit
ELISA Type:
Cell Based
€599

Description

Technical Manual

FKHRL1 (Phospho-Ser253) Cell-Based ELISA Kit

The FKHRL1 (Phospho-Ser253) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor FKHRL1 phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated FKHRL1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on FKHRL1 phosphorylation.

How does our FKHRL1 (Phospho-Ser253) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of FKHRL1 (Phospho-Ser253) concentration is achieved by an indirect ELISA format. In essence, FKHRL1 (Phospho-Ser253) is captured by FKHRL1 (Phospho-Ser253)-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 FKHRL1 (Phospho-Ser253) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated FKHRL1 can be used to normalize the RFU value for phosphorylated FKHRL1.

FKHRL1 (Phospho-Ser253) Fluorometric Cell-Based ELISA Kit -Information

Product Name:FKHRL1 (Phospho-Ser253) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00068
Description:The FKHRL1 (Phospho-Ser253) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor FKHRL1 (Phospho-Ser253) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated FKHRL1 (Phospho-Ser253) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on FOXO3 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: 2309, UniProt ID: O43524, OMIM #: 602681, Unigene #: Hs.220950
Format:Two 96-Well Plates
NCBI Gene Symbol:FOXO3
Sub Type:Phospho
Target Name:Phospho-FKHRL1 (Ser253)

Kit Principle

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

Kit components

Quantity

96-Well Black Cell CultureClear-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 Antibody110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody6 ml
Adhesive Plate Seals2 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 FKHRL1 (Phospho-Ser253) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total FKHRL1 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.

FKHRL1 (Phospho-Ser253) - Protein Information

UniProt Protein Function:FOXO3A: a transcription factors of the forkhead family. Contains 1 fork-head domain. May regulate apoptosis through control of genes necessary for cell death. Translocation of this gene with the MLL gene is associated with secondary acute leukemia.
UniProt Protein Details:

Protein type:Oncoprotein; DNA-binding; Transcription factor; Autophagy

Chromosomal Location of Human Ortholog: 6q21

Cellular Component: nucleoplasm; membrane; cytoplasm; cytosol; nucleus

Molecular Function:protein binding; DNA binding; chromatin DNA binding; sequence-specific DNA binding; transcription factor activity; protein kinase binding

Biological Process: epidermal growth factor receptor signaling pathway; transcription from RNA polymerase II promoter; phosphoinositide-mediated signaling; fibroblast growth factor receptor signaling pathway; DNA damage response, signal transduction by p53 class mediator; nerve growth factor receptor signaling pathway; initiation of primordial ovarian follicle growth; positive regulation of transcription, DNA-dependent; antral ovarian follicle growth; positive regulation of erythrocyte differentiation; negative regulation of transcription from RNA polymerase II promoter; glucose homeostasis; ovulation from ovarian follicle; oocyte maturation; regulation of translation; regulation of transcription from RNA polymerase II promoter; positive regulation of neuron apoptosis; innate immune response; positive regulation of transcription from RNA polymerase II promoter

NCBI Summary:This gene belongs to the forkhead family of transcription factors which are characterized by a distinct forkhead domain. This gene likely functions as a trigger for apoptosis through expression of genes necessary for cell death. Translocation of this gene with the MLL gene is associated with secondary acute leukemia. Alternatively spliced transcript variants encoding the same protein have been observed. [provided by RefSeq, Jul 2008]
UniProt Code:O43524
NCBI GenInfo Identifier:8134467
NCBI Gene ID:2309
NCBI Accession:O43524.1
UniProt Secondary Accession:O43524,O15171, Q5T2I7, Q9BZ04, B4DVZ6, E1P5E6,
UniProt Related Accession:O43524
Molecular Weight:
NCBI Full Name:Forkhead box protein O3
NCBI Synonym Full Names:forkhead box O3
NCBI Official Symbol:FOXO3  
NCBI Official Synonym Symbols:FOXO2; AF6q21; FKHRL1; FOXO3A; FKHRL1P2  
NCBI Protein Information:forkhead box protein O3; forkhead box O3A; forkhead in rhabdomyosarcoma-like 1; forkhead homolog (rhabdomyosarcoma) like 1; forkhead, Drosophila, homolog of, in rhabdomyosarcoma-like 1
UniProt Protein Name:Forkhead box protein O3
UniProt Synonym Protein Names:AF6q21 protein; Forkhead in rhabdomyosarcoma-like 1
Protein Family:Forkhead box protein
UniProt Gene Name:FOXO3  
UniProt Entry Name:FOXO3_HUMAN