CDC25B (Phospho-Ser323)Colorimetric Cell-Based ELISA Kit (CBCAB00399)

Product Name:	CDC25B (Phospho-Ser323)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00399
ELISA Type:	Cell-Based
Target:	CDC25B (Phospho-Ser323)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The CDC25B (Phospho-Ser323) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CDC25B protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CDC25B in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CDC25B phosphorylation.

Qualitative determination of CDC25B (Phospho-Ser323) concentration is achieved by an indirect ELISA format. In essence, CDC25B (Phospho-Ser323) is captured by CDC25B (Phospho-Ser323)-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: 994, UniProt ID: P30305, OMIM: 116949, Unigene: Hs.437705
Gene Symbol:	CDC25B
Sub Type:	Phospho

UniProt Protein Function:	CDC25B: 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; Nuclear receptor co-regulator; Protein phosphatase, dual-specificity; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 20p13 Cellular Component: nucleoplasm; spindle pole; centrosome; cytoplasm; cytosol Molecular Function:protein binding; protein tyrosine phosphatase activity; protein kinase binding Biological Process: positive regulation of cytokinesis; mitosis; cell division; positive regulation of protein kinase activity; positive regulation of cell proliferation; mitotic cell cycle; G2/M transition of mitotic cell cycle; positive regulation of mitotic cell cycle; protein amino acid phosphorylation; oocyte maturation; female meiosis I
NCBI Summary:	CDC25B is a member of the CDC25 family of phosphatases. CDC25B activates the cyclin dependent kinase CDC2 by removing two phosphate groups and it is required for entry into mitosis. CDC25B 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. Multiple transcript variants for this gene exist. [provided by RefSeq, Jul 2008]
UniProt Code:	P30305
NCBI GenInfo Identifier:	4757950
NCBI Gene ID:	994
NCBI Accession:	NP_004349.1
UniProt Secondary Accession:	P30305,O43551, Q13971, Q5JX77, Q6RSS1, Q9BRA6, D3DVY1 D3DVY2, D3DVY3, D3DVY4,
UniProt Related Accession:	P30305
Molecular Weight:	64,987 Da
NCBI Full Name:	M-phase inducer phosphatase 2 isoform 2
NCBI Synonym Full Names:	cell division cycle 25B
NCBI Official Symbol:	CDC25B
NCBI Protein Information:	M-phase inducer phosphatase 2; dual specificity phosphatase Cdc25B
UniProt Protein Name:	M-phase inducer phosphatase 2
UniProt Synonym Protein Names:	Dual specificity phosphatase Cdc25B
UniProt Gene Name:	CDC25B
UniProt Entry Name:	MPIP2_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 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.

Step	Procedure
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-CDC25B (Phospho-Ser323) Antibody, Anti-CDC25B 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.)

CDC25B (Phospho-Ser323)Colorimetric Cell-Based ELISA Kit (CBCAB00399)

Description