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TOB1 (Phospho-Ser164) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01692
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
Frequently bought together:

Description

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TOB1 (Phospho-Ser164)Colorimetric Cell-Based ELISA Kit

The TOB1 Phospho-Ser164 Colorimetric Cell-Based ELISA Kit is specifically designed for the accurate and reliable detection of TOB1 phosphorylated at serine 164 levels in cell lysates. This kit offers high sensitivity and specificity, ensuring precise and reproducible results for your research needs.TOB1, a known tumor suppressor gene, is involved in cell cycle regulation, proliferation, and apoptosis. Phosphorylation of TOB1 at serine 164 plays a crucial role in its function, making it a key marker for studying various cellular processes and signaling pathways.

By using the TOB1 Phospho-Ser164 Colorimetric Cell-Based ELISA Kit, researchers can gain valuable insights into the regulatory mechanisms of TOB1 phosphorylation and its impact on cellular functions. This kit is an essential tool for advancing research in cancer biology, cell signaling, and drug development. Elevate your research with the TOB1 Phospho-Ser164 Colorimetric Cell-Based ELISA Kit from Assay Genie.

Product Name:TOB1 (Phospho-Ser164) Colorimetric Cell-Based ELISA
Product Code:CBCAB01692
ELISA Type:Cell-Based
Target:TOB1 (Phospho-Ser164)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The TOB1 (Phospho-Ser164) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TOB1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated TOB1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on TOB1 phosphorylation.

Qualitative determination of TOB1 (Phospho-Ser164) concentration is achieved by an indirect ELISA format. In essence, TOB1 (Phospho-Ser164) is captured by TOB1 (Phospho-Ser164)-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: 10140, UniProt ID: P50616, OMIM: 605523, Unigene: Hs.709952/Hs.714409/Hs.714780
Gene Symbol:TOB1
Sub Type:Phospho
UniProt Protein Function:TOB1: Anti-proliferative protein that interacts with the erbB- 2 receptor tyrosine kinase. May physically and/or functionally interact with protein-tyrosine kinase receptors. Belongs to the BTG family.
UniProt Protein Details:

Protein type:Adaptor/scaffold; Cell cycle regulation

Chromosomal Location of Human Ortholog: 17q21

Cellular Component: CCR4-NOT complex

Molecular Function:protein binding; receptor tyrosine kinase binding; SH3/SH2 adaptor activity

Biological Process: negative regulation of cell proliferation; negative regulation of translation

NCBI Summary:This gene encodes a member of the transducer of erbB-2 /B-cell translocation gene protein family. Members of this family are anti-proliferative factors that have the potential to regulate cell growth. The encoded protein may function as a tumor suppressor. Alternate splicing results in multiple transcript variants. [provided by RefSeq, Aug 2011]
UniProt Code:P50616
NCBI GenInfo Identifier:1729989
NCBI Gene ID:10140
NCBI Accession:P50616.1
UniProt Secondary Accession:P50616,Q4KMQ0, B2R9T0, D3DTY3,
UniProt Related Accession:P50616
Molecular Weight:38,155 Da
NCBI Full Name:Protein Tob1
NCBI Synonym Full Names:transducer of ERBB2, 1
NCBI Official Symbol:TOB1  
NCBI Official Synonym Symbols:TOB; TROB; APRO5; APRO6; PIG49; TROB1  
NCBI Protein Information:protein Tob1
UniProt Protein Name:Protein Tob1
UniProt Synonym Protein Names:Transducer of erbB-2 1
Protein Family:Protein
UniProt Gene Name:TOB1  
UniProt Entry Name:TOB1_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-TOB1 (Phospho-Ser164) Antibody, Anti-TOB1 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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