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Breast Tumor Kinase (Phospho-Tyr447) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01369
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS
Product Name:Breast Tumor Kinase (Phospho-Tyr447) Colorimetric Cell-Based ELISA
Product Code:CBCAB01369
ELISA Type:Cell-Based
Target:Breast Tumor Kinase (Phospho-Tyr447)
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The Breast Tumor Kinase (Phospho-Tyr447) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Breast Tumor Kinase protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Breast Tumor Kinase in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Breast Tumor Kinase phosphorylation.

Qualitative determination of Breast Tumor Kinase (Phospho-Tyr447) concentration is achieved by an indirect ELISA format. In essence, Breast Tumor Kinase (Phospho-Tyr447) is captured by Breast Tumor Kinase (Phospho-Tyr447)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 5753, UniProt ID: Q13882, OMIM: 602004, Unigene: Hs.51133
Gene Symbol:PTK6
Sub Type:Phospho
UniProt Protein Function:Brk: a tyrosine kinase of the Src family. May function as an intracellular signal transducer in epithelial tissues. Very high level expression in colon and high levels in small intestine and prostate, and low levels in some fetal tissues. Selectively expressed in breast tumors and cell lines, and perhaps in colon and prostate cancers. Also found in melanocytes. Not expressed in heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. Overexpression in mammary cells leads to EGF sensitization and results in a partially transformed phenotype. Enhances anchorage-independent growth and responsiveness to EGF. RNAi reduces proliferation in breast cancer cells. Kinase-inactive mutant indicates tumor function may be independent of catalytic function.
UniProt Protein Details:

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

Chromosomal Location of Human Ortholog: 20q13.3

Cellular Component: nucleoplasm; ruffle; extrinsic to internal side of plasma membrane; cytoplasm; nucleus

Molecular Function:identical protein binding; protein binding; non-membrane spanning protein tyrosine kinase activity; ATP binding; receptor binding

Biological Process: cell migration; protein amino acid autophosphorylation; tyrosine phosphorylation of Stat3 protein; innate immune response; actin cytoskeleton organization and biogenesis; tyrosine phosphorylation of Stat5 protein; protein amino acid phosphorylation; transmembrane receptor protein tyrosine kinase signaling pathway; negative regulation of growth; regulation of cell proliferation

NCBI Summary:The protein encoded by this gene is a cytoplasmic nonreceptor protein kinase which may function as an intracellular signal transducer in epithelial tissues. Overexpression of this gene in mammary epithelial cells leads to sensitization of the cells to epidermal growth factor and results in a partially transformed phenotype. Expression of this gene has been detected at low levels in some breast tumors but not in normal breast tissue. The encoded protein has been shown to undergo autophosphorylation. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jan 2012]
UniProt Code:Q13882
NCBI GenInfo Identifier:8928302
NCBI Gene ID:5753
NCBI Accession:Q13882.1
UniProt Secondary Accession:Q13882,Q58F01, B2RCR3, B4DW46,
UniProt Related Accession:Q13882
Molecular Weight:451
NCBI Full Name:Protein-tyrosine kinase 6
NCBI Synonym Full Names:protein tyrosine kinase 6
NCBI Official Symbol:PTK6  
NCBI Official Synonym Symbols:BRK  
NCBI Protein Information:protein-tyrosine kinase 6; breast tumor kinase; protein-tyrosine kinase BRK; tyrosine-protein kinase BRK; PTK6 protein tyrosine kinase 6
UniProt Protein Name:Protein-tyrosine kinase 6
UniProt Synonym Protein Names:Breast tumor kinase; Tyrosine-protein kinase BRK
Protein Family:Protein-tyrosine kinase
UniProt Gene Name:PTK6  
UniProt Entry Name:PTK6_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-Breast Tumor Kinase (Phospho-Tyr447) Antibody, Anti-Breast Tumor Kinase 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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