ETK (Phospho-Tyr566) Fluorometric Cell-Based ELISA Kit

SKU:
FBCAB00046
Product type:
ELISA Kit
ELISA Type:
Cell Based
€599

Description

Technical Manual

ETK (Phospho-Tyr566) Cell-Based ELISA Kit

The ETK (Phospho-Tyr566) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor ETK phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated ETK in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on ETK phosphorylation.

How does our ETK (Phospho-Tyr566) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of ETK (Phospho-Tyr566) concentration is achieved by an indirect ELISA format. In essence, ETK (Phospho-Tyr566) is captured by ETK (Phospho-Tyr566)-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 ETK (Phospho-Tyr566) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated ETK can be used to normalize the RFU value for phosphorylated ETK.

ETK (Phospho-Tyr566) Fluorometric Cell-Based ELISA Kit -Information

Product Name:ETK (Phospho-Tyr566) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00046
Description:The ETK (Phospho-Tyr566) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor ETK (Phospho-Tyr566) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated ETK (Phospho-Tyr566) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on BMX phosphorylation.
Dynamic Range:> 5000 Cells
Detection Method:Fluorometric
Storage/Stability:4°C/6 Months
Reactivity:Human, Mouse
Assay Type:Cell-Based ELISA
Database Links:Gene ID: 660, UniProt ID: P51813, OMIM #: 300101, Unigene #: Hs.495731
Format:Two 96-Well Plates
NCBI Gene Symbol:BMX
Sub Type:Phospho
Target Name:Phospho-ETK (Tyr566)

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 ETK (Phospho-Tyr566) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total ETK 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.

ETK (Phospho-Tyr566) - Protein Information

UniProt Protein Function:Etk: a tyrosine kinase of the Tec family. Activity is required for IL6-induced differentiation. May play a role in the growth and differentiation of hematopoietic cells. May be involved in signal transduction in endocardial and arterial endothelial cells.
UniProt Protein Details:

Protein type:Protein kinase, TK; Kinase, protein; EC 2.7.10.2; Protein kinase, tyrosine (non-receptor); TK group; Tec family

Chromosomal Location of Human Ortholog: Xp22.2

Cellular Component: cytosol; extrinsic to internal side of plasma membrane

Molecular Function:ATP binding; metal ion binding; non-membrane spanning protein tyrosine kinase activity; protein binding; protein-tyrosine kinase activity; receptor binding; signal transducer activity

Biological Process: apoptosis; cell structure disassembly during apoptosis; innate immune response; mesoderm development; NK T cell differentiation; programmed cell death; protein amino acid autophosphorylation; protein amino acid phosphorylation; regulation of cell proliferation; signal transduction; transmembrane receptor protein tyrosine kinase signaling pathway

NCBI Summary:This gene encodes a non-receptor tyrosine kinase belonging to the Tec kinase family. The protein contains a PH-like domain, which mediates membrane targeting by binding to phosphatidylinositol 3,4,5-triphosphate (PIP3), and a SH2 domain that binds to tyrosine-phosphorylated proteins and functions in signal transduction. The protein is implicated in several signal transduction pathways including the Stat pathway, and regulates differentiation and tumorigenicity of several types of cancer cells. Alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Mar 2016]
UniProt Code:P51813
NCBI GenInfo Identifier:1705489
NCBI Gene ID:660
NCBI Accession:P51813.1
UniProt Secondary Accession:P51813,O60564, Q12871, A6NIH9,
UniProt Related Accession:P51813
Molecular Weight:
NCBI Full Name:Cytoplasmic tyrosine-protein kinase BMX
NCBI Synonym Full Names:BMX non-receptor tyrosine kinase
NCBI Official Symbol:BMX  
NCBI Official Synonym Symbols:ETK; PSCTK2; PSCTK3  
NCBI Protein Information:cytoplasmic tyrosine-protein kinase BMX
UniProt Protein Name:Cytoplasmic tyrosine-protein kinase BMX
UniProt Synonym Protein Names:Bone marrow tyrosine kinase gene in chromosome X protein; Epithelial and endothelial tyrosine kinase; ETK; NTK38
Protein Family:Cytoplasmic tyrosine-protein kinase
UniProt Gene Name:BMX  
UniProt Entry Name:BMX_HUMAN