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CDC25C (Phospho-Thr48) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00431
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Cell Cycle
Reactivity:
Human
Detection Method:
Colorimetric
€599
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Description

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CDC25C (Phospho-Thr48)Colorimetric Cell-Based ELISA Kit

The CDC25C Phospho-Thr48 Colorimetric Cell-Based ELISA Kit is a powerful tool for detecting phosphorylated CDC25C at threonine 48 in cell samples. This kit offers high sensitivity and precision, allowing for accurate and reproducible results in a variety of research applications.CDC25C is a key regulator of cell cycle progression, influencing checkpoints and ensuring proper cell division. Phosphorylation of CDC25C at threonine 48 is a critical modification that regulates its activity, making it an important target for studying cell cycle control and potential therapeutic interventions.

This kit is easy to use and delivers rapid results, making it suitable for high-throughput screening and detailed mechanistic studies. By accurately measuring phosphorylated CDC25C levels, researchers can gain valuable insights into cell cycle regulation and identify novel pathways for therapeutic development.

Product Name:CDC25C (Phospho-Thr48) Colorimetric Cell-Based ELISA
Product Code:CBCAB00431
ELISA Type:Cell-Based
Target:CDC25C (Phospho-Thr48)
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The CDC25C (Phospho-Thr48) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CDC25C protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CDC25C in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CDC25C phosphorylation.

Qualitative determination of CDC25C (Phospho-Thr48) concentration is achieved by an indirect ELISA format. In essence, CDC25C (Phospho-Thr48) is captured by CDC25C (Phospho-Thr48)-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: 995, UniProt ID: P30307, OMIM: 157680, Unigene: Hs.656
Gene Symbol:CDC25A
Sub Type:Phospho
UniProt Protein Function:CDC25C: a member of the MPI phosphatase family. Activates the cyclin dependent kinase CDC2 by removing two phosphate groups and it is required for entry into mitosis. Shuttles between the nucleus and the cytoplasm due to nuclear localization and nuclear export signals. The protein is nuclear in the M and G1 phases of the cell cycle and moves to the cytoplasm during S and G2. CDC25B has oncogenic properties, although its role in tumor formation has not been determined. At least four transcript variants for this gene exist. Three splice variant isoforms have been described.
UniProt Protein Details:

Protein type:EC 3.1.3.48; Motility/polarity/chemotaxis; Protein phosphatase, dual-specificity

Chromosomal Location of Human Ortholog: 5q31

Cellular Component: cytosol; nucleoplasm; nucleus

Molecular Function:phosphoprotein phosphatase activity; protein binding; protein kinase binding; WW domain binding

Biological Process: cell proliferation; DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest; DNA replication; G2/M transition of mitotic cell cycle; regulation of cell cycle; regulation of cyclin-dependent protein kinase activity; regulation of mitosis

NCBI Summary:This gene encodes a conserved protein that plays a key role in the regulation of cell division. The encoded protein directs dephosphorylation of cyclin B-bound CDC2 and triggers entry into mitosis. It also suppresses p53-induced growth arrest. Multiple alternatively spliced transcript variants of this gene have been described. [provided by RefSeq, Dec 2015]
UniProt Code:P30307
NCBI GenInfo Identifier:116242631
NCBI Gene ID:995
NCBI Accession:P30307.2
UniProt Secondary Accession:P30307,Q96PL3, Q9H168, Q9H2E8, Q9H2E9, Q9H2F1, D3DQB8
UniProt Related Accession:P30307
Molecular Weight:45,608 Da
NCBI Full Name:M-phase inducer phosphatase 3
NCBI Synonym Full Names:cell division cycle 25C
NCBI Official Symbol:CDC25C  
NCBI Official Synonym Symbols:CDC25; PPP1R60  
NCBI Protein Information:M-phase inducer phosphatase 3
UniProt Protein Name:M-phase inducer phosphatase 3
UniProt Synonym Protein Names:Dual specificity phosphatase Cdc25C
UniProt Gene Name:CDC25C  
UniProt Entry Name:MPIP3_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-CDC25C (Phospho-Thr48) Antibody, Anti-CDC25C 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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