CDC6 Colorimetric Cell-Based ELISA Kit (CBCAB00574)

Product Name:	CDC6 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00574
ELISA Type:	Cell-Based
Target:	CDC6
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The CDC6 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CDC6 protein expression profile in cells. The kit can be used for measuring the relative amounts of CDC6 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on CDC6.

Qualitative determination of CDC6 concentration is achieved by an indirect ELISA format. In essence, CDC6 is captured by CDC6-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: 990, UniProt ID: Q99741, OMIM: 602627, Unigene: Hs.405958
Gene Symbol:	CDC6
Sub Type:	None

UniProt Protein Function:	CDC6: Involved in the initiation of DNA replication. Also participates in checkpoint controls that ensure DNA replication is completed before mitosis is initiated. Interacts with PCNA, ORC1L, cyclin-CDK and HUWE1. Belongs to the CDC6/cdc18 family.
UniProt Protein Details:	Protein type:Cell cycle regulation Chromosomal Location of Human Ortholog: 17q21.3 Cellular Component: cytoplasm; cytosol; Golgi apparatus; nucleoplasm; nucleus; spindle midzone; spindle pole Molecular Function:kinase binding; nucleotide binding; protein binding Biological Process: DNA replication; DNA replication checkpoint; G1/S transition of mitotic cell cycle; G1/S-specific transcription in mitotic cell cycle; negative regulation of cell proliferation; negative regulation of DNA replication; positive regulation of chromosome segregation; positive regulation of cytokinesis; regulation of cyclin-dependent protein kinase activity; regulation of mitotic metaphase/anaphase transition; traversing start control point of mitotic cell cycle Disease: Meier-gorlin Syndrome 5
NCBI Summary:	The protein encoded by this gene is highly similar to Saccharomyces cerevisiae Cdc6, a protein essential for the initiation of DNA replication. This protein functions as a regulator at the early steps of DNA replication. It localizes in cell nucleus during cell cyle G1, but translocates to the cytoplasm at the start of S phase. The subcellular translocation of this protein during cell cyle is regulated through its phosphorylation by Cdks. Transcription of this protein was reported to be regulated in response to mitogenic signals through transcriptional control mechanism involving E2F proteins. [provided by RefSeq, Jul 2008]
UniProt Code:	Q99741
NCBI GenInfo Identifier:	50400620
NCBI Gene ID:	990
NCBI Accession:	Q99741.1
UniProt Secondary Accession:	Q99741,Q8TB30,
UniProt Related Accession:	Q99741
Molecular Weight:	62,720 Da
NCBI Full Name:	Cell division control protein 6 homolog
NCBI Synonym Full Names:	cell division cycle 6
NCBI Official Symbol:	CDC6
NCBI Official Synonym Symbols:	CDC18L; HsCDC6; HsCDC18
NCBI Protein Information:	cell division control protein 6 homolog
UniProt Protein Name:	Cell division control protein 6 homolog
UniProt Synonym Protein Names:	CDC6-related protein; Cdc18-related protein; HsCdc18; p62(cdc6); HsCDC6
Protein Family:	Cell division control protein
UniProt Gene Name:	CDC6
UniProt Entry Name:	CDC6_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-CDC6 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.)