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14-3-3 theta/tau (Phospho-Ser232) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00436
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
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Description

system_update_altDatasheetMSDS

14-3-3 theta/tau (Phospho-Ser232)Colorimetric Cell-Based ELISA Kit

The 14-3-3 Theta (Tau) Phospho-Ser232 Colorimetric Cell-Based ELISA Kit is a powerful tool for the accurate quantification of phosphorylated 14-3-3 Theta (Tau) protein levels in cell lysates. This kit is highly sensitive and specific, allowing for precise and reliable results for various research applications.The 14-3-3 Theta (Tau) protein is a key regulator of multiple cellular processes, including cell cycle progression, apoptosis, and signal transduction.

Phosphorylation at serine 232 is critical for its function and can play a role in various diseases such as cancer, neurodegenerative disorders, and metabolic diseases. This ELISA kit enables researchers to study the phosphorylation status of 14-3-3 Theta (Tau) protein and its implications in disease pathology and potential therapeutic strategies.

Product Name:14-3-3 theta/tau (Phospho-Ser232) Colorimetric Cell-Based ELISA
Product Code:CBCAB00436
ELISA Type:Cell-Based
Target:14-3-3 theta/tau (Phospho-Ser232)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The 14-3-3 theta/tau (Phospho-Ser232) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect 14-3-3 theta/tau protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated 14-3-3 theta/tau in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on 14-3-3 theta/tau phosphorylation.

Qualitative determination of 14-3-3 theta/tau (Phospho-Ser232) concentration is achieved by an indirect ELISA format. In essence, 14-3-3 theta/tau (Phospho-Ser232) is captured by 14-3-3 theta/tau (Phospho-Ser232)-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: 5350, UniProt ID: P27348, OMIM: 172405/609909, Unigene: Hs.170839
Gene Symbol:YWHAQ
Sub Type:Phospho
UniProt Protein Function:14-3-3 theta: a protein of the 14-3-3 family of proteins which mediate signal transduction by binding to phosphoserine-containing proteins. A multifunctional regulator of the cell signaling processes. Associates with c-BCR and BCR-Abl.
UniProt Protein Details:

Protein type:Adaptor/scaffold

Chromosomal Location of Human Ortholog: 2p25.1

Cellular Component: cytoplasm; cytoplasmic vesicle membrane; cytosol; focal adhesion; membrane; mitochondrion; protein complex

Molecular Function:protein binding; protein C-terminus binding; protein domain specific binding; protein N-terminus binding

Biological Process: apoptosis; gene expression; negative regulation of transcription, DNA-dependent; programmed cell death; small GTPase mediated signal transduction; substantia nigra development; transcription initiation from RNA polymerase II promoter

NCBI Summary:This gene product belongs to the 14-3-3 family of proteins which mediate signal transduction by binding to phosphoserine-containing proteins. This highly conserved protein family is found in both plants and mammals, and this protein is 99% identical to the mouse and rat orthologs. This gene is upregulated in patients with amyotrophic lateral sclerosis. It contains in its 5' UTR a 6 bp tandem repeat sequence which is polymorphic, however, there is no correlation between the repeat number and the disease. [provided by RefSeq, Jul 2008]
UniProt Code:P27348
NCBI GenInfo Identifier:112690
NCBI Gene ID:10971
NCBI Accession:P27348.1
UniProt Secondary Accession:P27348,Q567U5, Q5TZU8, Q9UP48, D6W4Z5,
UniProt Related Accession:P27348
Molecular Weight:
NCBI Full Name:14-3-3 protein theta
NCBI Synonym Full Names:tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta
NCBI Official Symbol:YWHAQ  
NCBI Official Synonym Symbols:1C5; HS1; 14-3-3  
NCBI Protein Information:14-3-3 protein theta
UniProt Protein Name:14-3-3 protein theta
UniProt Synonym Protein Names:14-3-3 protein T-cell; 14-3-3 protein tau; Protein HS1
Protein Family:14-3-3 protein
UniProt Gene Name:YWHAQ  
UniProt Entry Name:1433T_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-14-3-3 theta/tau (Phospho-Ser232) Antibody, Anti-14-3-3 theta/tau 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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