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

SKU:
FBCAB00040
Product type:
ELISA Kit
ELISA Type:
Cell Based
€599

Description

Technical Manual

CDC25C (Phospho-Thr48) Cell-Based ELISA Kit

The CDC25C (Phospho-Thr48) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor CDC25C 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.

How does our CDC25C (Phospho-Thr48) Fluorometric Cell-Based ELISA Kit work?

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 (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 CDC25C (Phospho-Thr48) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated CDC25C can be used to normalize the RFU value for phosphorylated CDC25C.

CDC25C (Phospho-Thr48) Fluorometric Cell-Based ELISA Kit -Information

Product Name:CDC25C (Phospho-Thr48) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00040
Description:The CDC25C (Phospho-Thr48) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor CDC25C (Phospho-Thr48) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CDC25C (Phospho-Thr48) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on CDC25A phosphorylation.
Dynamic Range:> 5000 Cells
Detection Method:Fluorometric
Storage/Stability:4°C/6 Months
Reactivity:Human
Assay Type:Cell-Based ELISA
Database Links:Gene ID: 995, UniProt ID: P30307, OMIM #: 157680, Unigene #: Hs.656
Format:Two 96-Well Plates
NCBI Gene Symbol:CDC25A
Sub Type:Phospho
Target Name:Phospho-CDC25C (Thr48)

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 CDC25C (Phospho-Thr48) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total CDC25C 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.

CDC25C (Phospho-Thr48) - Protein Information

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