null

WWOX (Phospho-Tyr33) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01668
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Cell Death
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

system_update_altDatasheetMSDS

WWOX (Phospho-Tyr33)Colorimetric Cell-Based ELISA Kit

The Wwox Phospho-Tyr33 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool designed for the accurate and sensitive detection of Wwox phosphorylated at Tyr33 in cell lysates and tissue samples. This kit offers high specificity and reproducibility, providing researchers with reliable results for their studies.Wwox, also known as WW domain-containing oxidoreductase, is a tumor suppressor protein that plays a critical role in various cellular processes, including cell growth, apoptosis, and DNA repair.

Phosphorylation of Wwox at Tyr33 has been linked to cancer development and progression, making it a valuable biomarker for cancer research and drug development.With the Wwox Phospho-Tyr33 Colorimetric Cell-Based ELISA Kit, researchers can gain valuable insights into the role of Wwox phosphorylation in cancer and other diseases, paving the way for innovative therapeutic strategies and personalized medicine approaches.

Product Name:WWOX (Phospho-Tyr33) Colorimetric Cell-Based ELISA
Product Code:CBCAB01668
ELISA Type:Cell-Based
Target:WWOX (Phospho-Tyr33)
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The WWOX (Phospho-Tyr33) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect WWOX protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated WWOX in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on WWOX phosphorylation.

Qualitative determination of WWOX (Phospho-Tyr33) concentration is achieved by an indirect ELISA format. In essence, WWOX (Phospho-Tyr33) is captured by WWOX (Phospho-Tyr33)-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: 51741, UniProt ID: Q96KM3, OMIM: 133239/605131, Unigene: Hs.461453
Gene Symbol:WWOX
Sub Type:Phospho
UniProt Protein Function:WWOX: an enzyme which contains 2 WW domains and a short-chain dehydrogenase/reductase domain (SRD). Expressed at high levels in hormonally regulated tissues such as testis, ovary, and prostate. Its SRD domain suggest a role for this gene in steroid metabolism. Shown to be an essential mediator of tumor necrosis factor-alpha-induced apoptosis in the mouse. May play a similar role in the human. May function as a suppressor of tumor growth. Alternative splicing of this gene generates 7 isoforms.
UniProt Protein Details:

Protein type:EC 1.1.1.-; Apoptosis; Oxidoreductase; Tumor suppressor

Chromosomal Location of Human Ortholog: 16q23

Cellular Component: cytoplasm; cytosol; Golgi apparatus; microvillus; mitochondrion; nucleus; plasma membrane

Molecular Function:coenzyme binding; cofactor binding; enzyme binding; oxidoreductase activity; protein binding; protein dimerization activity

Biological Process: negative regulation of Wnt receptor signaling pathway; osteoblast differentiation; positive regulation of transcription from RNA polymerase II promoter; skeletal morphogenesis; steroid metabolic process; Wnt receptor signaling pathway

Disease: Epileptic Encephalopathy, Early Infantile, 28

NCBI Summary:This gene encodes a member of the short-chain dehydrogenases/reductases (SDR) protein family. This gene spans the FRA16D common chromosomal fragile site and appears to function as a tumor suppressor gene. Expression of the encoded protein is able to induce apoptosis, while defects in this gene are associated with multiple types of cancer. Disruption of this gene is also associated with autosomal recessive spinocerebellar ataxia 12. Disruption of a similar gene in mouse results in impaired steroidogenesis, additionally suggesting a metabolic function for the protein. Alternative splicing results in multiple transcript variants. [provided by RefSeq, May 2014]
UniProt Code:Q96KM3
NCBI GenInfo Identifier:635172870
NCBI Gene ID:51741
NCBI Accession:NP_001278926.1
UniProt Secondary Accession:Q96KM3,Q5MYT5, Q96KM3, Q96RF2, Q9BTT8, Q9NPC9, Q9NRF4 Q9NRF5, Q9NRF6, Q9NRK1, Q9NZC5, A8K323,
UniProt Related Accession:Q9NZC7
Molecular Weight:23,868 Da
NCBI Full Name:WW domain-containing oxidoreductase isoform 4
NCBI Synonym Full Names:WW domain containing oxidoreductase
NCBI Official Symbol:WWOX  
NCBI Official Synonym Symbols:FOR; WOX1; EIEE28; FRA16D; SCAR12; HHCMA56; PRO0128; SDR41C1; D16S432E  
NCBI Protein Information:WW domain-containing oxidoreductase
UniProt Protein Name:WW domain-containing oxidoreductase
UniProt Synonym Protein Names:Fragile site FRA16D oxidoreductase; Short chain dehydrogenase/reductase family 41C member 1
Protein Family:WW domain-containing oxidoreductase
UniProt Gene Name:WWOX  
UniProt Entry Name:WWOX_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-WWOX (Phospho-Tyr33) Antibody, Anti-WWOX 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.)

Related Products