Catenin-beta (Phospho-Tyr489) Fluorometric Cell-Based ELISA Kit

SKU:
FBCAB00086
Product type:
ELISA Kit
ELISA Type:
Cell Based
€599

Description

Technical Manual

Catenin-beta (Phospho-Tyr489) Cell-Based ELISA Kit

The Catenin-beta (Phospho-Tyr489) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor Catenin-beta phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Catenin-beta in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Catenin-beta phosphorylation.

How does our Catenin-beta (Phospho-Tyr489) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of Catenin-beta (Phospho-Tyr489) concentration is achieved by an indirect ELISA format. In essence, Catenin-beta (Phospho-Tyr489) is captured by Catenin-beta (Phospho-Tyr489)-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 Catenin-beta (Phospho-Tyr489) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated Catenin-beta can be used to normalize the RFU value for phosphorylated Catenin-beta.

Catenin-beta (Phospho-Tyr489) Fluorometric Cell-Based ELISA Kit -Information

Product Name:Catenin-beta (Phospho-Tyr489) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00086
Description:The Catenin-beta (Phospho-Tyr489) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor Catenin-? (Phospho-Tyr489) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Catenin-? (Phospho-Tyr489) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on CTNNB1 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: 1499, UniProt ID: P35222, OMIM #: 116806/132600/155255, Unigene #: Hs.476018
Format:Two 96-Well Plates
NCBI Gene Symbol:CTNNB1
Sub Type:Phospho
Target Name:Phospho-Catenin-? (Tyr489)

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 Catenin-beta (Phospho-Tyr489) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total Catenin-beta 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.

Catenin-beta (Phospho-Tyr489) - Protein Information

UniProt Protein Function:CTNNB1: a regulator of cell adhesion and a key downstream effector in the Wnt signaling pathway. Implicated early embryonic development and tumorigenesis. Phosphorylated and destabilized by CK1 and GSK-3beta. Stabilized cytoplasmic beta-catenin is a hallmark of a variety of cancers. Stabilized beta-catenin translocates to the nucleus, where it acts as a transcriptional activator of T-cell factor (TCF)-regulated genes. Interacts with the PDZ domain of TAX1BP3, inhibiting its transcriptional activity. Two alternatively spliced human isoforms have been described.
UniProt Protein Details:

Protein type:Transcription factor; Motility/polarity/chemotaxis; Cell adhesion; Oncoprotein; Nuclear receptor co-regulator; Actin-binding

Chromosomal Location of Human Ortholog: 3p21

Cellular Component: centrosome; basolateral plasma membrane; intercellular junction; fascia adherens; cytosol; beta-catenin destruction complex; transcription factor complex; cell-cell adherens junction; membrane; lamellipodium; perinuclear region of cytoplasm; cytoplasm; synapse; dendritic shaft; lateral plasma membrane; spindle pole; focal adhesion; tight junction; catenin complex; cell cortex; Z disc; nucleoplasm; adherens junction; microvillus membrane; apical part of cell; plasma membrane; nucleus; cell junction

Molecular Function:protein C-terminus binding; transcription coactivator activity; transcription factor binding; protein phosphatase binding; ionotropic glutamate receptor binding; protein binding; signal transducer activity; enzyme binding; androgen receptor binding; cadherin binding; double-stranded DNA binding; protein complex binding; estrogen receptor binding; nitric-oxide synthase binding; SMAD binding; transcription factor activity; kinase binding; alpha-catenin binding; nuclear hormone receptor binding

Biological Process: regulation of myelination; regulation of centriole-centriole cohesion; positive regulation of apoptosis; protein heterooligomerization; positive regulation of transcription, DNA-dependent; regulation of fibroblast proliferation; cell maturation; negative regulation of chondrocyte differentiation; T cell differentiation in the thymus; positive regulation of fibroblast growth factor receptor signaling pathway; osteoclast differentiation; Wnt receptor signaling pathway through beta-catenin; cell-cell adhesion; positive regulation of endothelial cell differentiation; regulation of cell fate specification; embryonic foregut morphogenesis; positive regulation of mesenchymal cell proliferation; male genitalia development; ectoderm development; synapse organization and biogenesis; cell adhesion; bone resorption; response to drug; positive regulation of neuroblast proliferation; positive regulation of I-kappaB kinase/NF-kappaB cascade; regulation of smooth muscle cell proliferation; transcription, DNA-dependent; hair cell differentiation; negative regulation of protein sumoylation; patterning of blood vessels; genitalia morphogenesis; muscle cell differentiation; midgut development; smooth muscle cell differentiation; positive regulation of transcription from RNA polymerase II promoter; embryonic digit morphogenesis; negative regulation of transcription, DNA-dependent; oocyte development; embryonic forelimb morphogenesis; negative regulation of osteoclast differentiation; glial cell fate determination; endodermal cell fate commitment; apoptosis; cell-matrix adhesion; neuron migration; dorsal/ventral axis specification; cell fate specification; positive regulation of histone H3-K4 methylation; negative regulation of transcription from RNA polymerase II promoter; embryonic hindlimb morphogenesis; response to estradiol stimulus; negative regulation of cell proliferation; central nervous system vasculogenesis; positive regulation of MAPKKK cascade; pancreas development; positive regulation of interferon type I production; fallopian tube development; proximal/distal pattern formation; layer formation in the cerebral cortex; negative regulation of mitotic cell cycle, embryonic; cell structure disassembly during apoptosis; Wnt receptor signaling pathway; hair follicle morphogenesis; thymus development; in utero embryonic development; regulation of T cell proliferation; neural plate development; stem cell maintenance; embryonic axis specification; synaptic vesicle transport; gastrulation with mouth forming second; liver development; regulation of angiogenesis; odontogenesis of dentine-containing teeth; myoblast differentiation; negative regulation of oligodendrocyte differentiation; Schwann cell proliferation; positive regulation of osteoblast differentiation; response to cadmium ion; ureteric bud branching; response to cytokine stimulus; androgen receptor signaling pathway; epithelial to mesenchymal transition; positive regulation of muscle cell differentiation; embryonic heart tube development; innate immune response; lens morphogenesis in camera-type eye; anterior/posterior axis specification

Disease: Pilomatrixoma; Mental Retardation, Autosomal Dominant 19; Ovarian Cancer; Colorectal Cancer; Hepatocellular Carcinoma

NCBI Summary:The protein encoded by this gene is part of a complex of proteins that constitute adherens junctions (AJs). AJs are necessary for the creation and maintenance of epithelial cell layers by regulating cell growth and adhesion between cells. The encoded protein also anchors the actin cytoskeleton and may be responsible for transmitting the contact inhibition signal that causes cells to stop dividing once the epithelial sheet is complete. Finally, this protein binds to the product of the APC gene, which is mutated in adenomatous polyposis of the colon. Mutations in this gene are a cause of colorectal cancer (CRC), pilomatrixoma (PTR), medulloblastoma (MDB), and ovarian cancer. Three transcript variants encoding the same protein have been found for this gene.[provided by RefSeq, Oct 2009]
UniProt Code:P35222
NCBI GenInfo Identifier:461854
NCBI Gene ID:1499
NCBI Accession:P35222.1
UniProt Secondary Accession:P35222,Q8NEW9, Q8NI94, Q9H391, A8K1L7,
UniProt Related Accession:P35222
Molecular Weight:
NCBI Full Name:Catenin beta-1
NCBI Synonym Full Names:catenin (cadherin-associated protein), beta 1, 88kDa
NCBI Official Symbol:CTNNB1  
NCBI Official Synonym Symbols:CTNNB; MRD19; armadillo  
NCBI Protein Information:catenin beta-1
UniProt Protein Name:Catenin beta-1
UniProt Synonym Protein Names:Beta-catenin
Protein Family:Catenin
UniProt Gene Name:CTNNB1  
UniProt Entry Name:CTNB1_HUMAN