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ETS1 (Phospho-Thr38) Fluorometric Cell-Based ELISA Kit

SKU:
FBCAB00027
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Mouse
Rat
Detection Method:
Fluorometric
€599
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Description

Technical Manual

ETS1 (Phospho-Thr38) Cell-Based ELISA Kit

The ETS1 (Phospho-Thr38) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor ETS1 phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated ETS1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on ETS1 phosphorylation.

How does our ETS1 (Phospho-Thr38) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of ETS1 (Phospho-Thr38) concentration is achieved by an indirect ELISA format. In essence, ETS1 (Phospho-Thr38) is captured by ETS1 (Phospho-Thr38)-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 ETS1 (Phospho-Thr38) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated ETS1 can be used to normalize the RFU value for phosphorylated ETS1.

ETS1 (Phospho-Thr38) Fluorometric Cell-Based ELISA Kit -Information

Product Name:ETS1 (Phospho-Thr38) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00027
Description:The ETS1 (Phospho-Thr38) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor ETS1 (Phospho-Thr38) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated ETS1 (Phospho-Thr38) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on ETS1 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: 2113, UniProt ID: P14921, OMIM #: 164720, Unigene #: Hs.369438
Format:Two 96-Well Plates
NCBI Gene Symbol:ETS1
Sub Type:Phospho
Target Name:Phospho-ETS1 (Thr38)

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 ETS1 (Phospho-Thr38) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total ETS1 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.

ETS1 (Phospho-Thr38) - Protein Information

UniProt Protein Function:Ets-1: a proto-oncogenic transcription factor homologous to the v-ets erythroblastosis virus oncogene. The DNA binding activity of Ets1 is controlled by kinases and transcription factors. It contributes to the regulation of cellular differentiation in hematopoietic cells. Ets1 promotes invasive behavior in endothelial cells, vascular smooth muscle cells and epithelial cancer cells. Regulates the expression of MMP1, MMP3, MMP9 and uPA as well as of VEGF and VEGFR gene expression. Two alternatively spliced isoforms have been described.
UniProt Protein Details:

Protein type:DNA-binding; Motility/polarity/chemotaxis; Oncoprotein; Transcription factor

Chromosomal Location of Human Ortholog: 11q23.3

Cellular Component: nucleoplasm; nucleus

Molecular Function:DNA binding; identical protein binding; protein binding; transcription factor activity

Biological Process: cell motility involved in cell locomotion; immune response; negative regulation of cell proliferation; PML body organization and biogenesis; positive regulation of cell motility; positive regulation of erythrocyte differentiation; positive regulation of inflammatory response; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; regulation of angiogenesis

NCBI Summary:This gene encodes a member of the ETS family of transcription factors, which are defined by the presence of a conserved ETS DNA-binding domain that recognizes the core consensus DNA sequence GGAA/T in target genes. These proteins function either as transcriptional activators or repressors of numerous genes, and are involved in stem cell development, cell senescence and death, and tumorigenesis. Alternatively spliced transcript variants encoding different isoforms have been described for this gene.[provided by RefSeq, Jul 2011]
UniProt Code:P14921
NCBI GenInfo Identifier:119641
NCBI Gene ID:2113
NCBI Accession:P14921.1
UniProt Secondary Accession:P14921,Q14278, Q16080, Q6N087, Q96AC5, A9UL17, F5GYX9
UniProt Related Accession:P14921
Molecular Weight:30,860 Da
NCBI Full Name:Protein C-ets-1
NCBI Synonym Full Names:ETS proto-oncogene 1, transcription factor
NCBI Official Symbol:ETS1  
NCBI Official Synonym Symbols:p54; ETS-1; EWSR2; c-ets-1  
NCBI Protein Information:protein C-ets-1
UniProt Protein Name:Protein C-ets-1
UniProt Synonym Protein Names:p54
Protein Family:Protein
UniProt Gene Name:ETS1  
UniProt Entry Name:ETS1_HUMAN

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