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ETK (Phospho-Tyr566) Colorimetric Cell-Based ELISA Kit (CBCAB00438)

SKU:
CBCAB00438
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Cell Death
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€960

Description

system_update_altDatasheetMSDS

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

The ETK Phospho-Tyr566 Colorimetric Cell-Based ELISA Kit is a powerful tool for detecting phosphorylation events in tyrosine 566 of specific proteins in cell-based assays. This kit provides highly sensitive and accurate measurements of phospho-Tyr566 levels, making it perfect for researchers studying protein phosphorylation and signal transduction pathways.The phosphorylation at tyrosine 566 is known to play a critical role in various cellular processes, including cell growth, differentiation, and migration. By accurately measuring this specific phosphorylation event, researchers can gain valuable insights into the molecular mechanisms underlying these processes and develop potential therapeutic targets for various diseases.

With its easy-to-use protocol, high specificity, and reliable results, the ETK Phospho-Tyr566 Colorimetric Cell-Based ELISA Kit is an essential tool for any laboratory involved in studying protein phosphorylation dynamics and signaling pathways. Trust Assay Genie to provide you with the cutting-edge tools you need for your research endeavors.

Product Name:ETK (Phospho-Tyr566) Colorimetric Cell-Based ELISA
Product Code:CBCAB00438
ELISA Type:Cell-Based
Target:ETK (Phospho-Tyr566)
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The ETK (Phospho-Tyr566) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ETK protein 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.

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 antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. 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 absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 660, UniProt ID: P51813, OMIM: 300101, Unigene: Hs.495731
Gene Symbol:BMX
Sub Type:Phospho
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
ComponentQuantity
96-Well Cell Culture Clear-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 Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 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)
  • Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. 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 five minutes each time with gentle shaking on the orbital shaker. 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 5 minutes each time.
9.Add 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 5 minutes each time.
11.Add 50 µL of 1x primary antibodies Anti-ETK (Phospho-Tyr566) Antibody, Anti-ETK Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover 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 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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