Cyclin D1 (Phospho-Thr286)Colorimetric Cell-Based ELISA Kit (CBCAB00407)

Product Name:	Cyclin D1 (Phospho-Thr286)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00407
ELISA Type:	Cell-Based
Target:	Cyclin D1 (Phospho-Thr286)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Cyclin D1 (Phospho-Thr286) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Cyclin D1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Cyclin D1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Cyclin D1 phosphorylation.

Qualitative determination of Cyclin D1 (Phospho-Thr286) concentration is achieved by an indirect ELISA format. In essence, Cyclin D1 (Phospho-Thr286) is captured by Cyclin D1 (Phospho-Thr286)-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: 595, UniProt ID: P24385, OMIM: 168461/254500, Unigene: Hs.523852
Gene Symbol:	CCND1
Sub Type:	Phospho

UniProt Protein Function:	CCND1: a member of the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle. Cyclins function as regulators of CDK kinases. Forms a complex with and functions as a regulatory subunit of CDK4 or CDK6, whose activity is required for cell cycle G1/S transition. This protein has been shown to interact with tumor suppressor protein Rb and the expression of this gene is regulated positively by Rb.
UniProt Protein Details:	Protein type:Activator; Cell cycle regulation; Nuclear receptor co-regulator; Oncoprotein Chromosomal Location of Human Ortholog: 11q13.3 Cellular Component: cyclin-dependent protein kinase holoenzyme complex; cytosol; intracellular; nucleoplasm; nucleus; transcriptional repressor complex Molecular Function:enzyme binding; histone deacetylase binding; protein binding; protein kinase binding; transcription corepressor activity; transcription factor binding Biological Process: G1 DNA damage checkpoint; G1/S transition of mitotic cell cycle; negative regulation of transcription from RNA polymerase II promoter; positive regulation of cell cycle; positive regulation of cyclin-dependent protein kinase activity; positive regulation of protein amino acid phosphorylation; response to DNA damage stimulus; response to drug Disease: Myeloma, Multiple; Von Hippel-lindau Syndrome
NCBI Summary:	The protein encoded by this gene belongs to the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance throughout the cell cycle. Cyclins function as regulators of CDK kinases. Different cyclins exhibit distinct expression and degradation patterns which contribute to the temporal coordination of each mitotic event. This cyclin forms a complex with and functions as a regulatory subunit of CDK4 or CDK6, whose activity is required for cell cycle G1/S transition. This protein has been shown to interact with tumor suppressor protein Rb and the expression of this gene is regulated positively by Rb. Mutations, amplification and overexpression of this gene, which alters cell cycle progression, are observed frequently in a variety of tumors and may contribute to tumorigenesis. [provided by RefSeq, Jul 2008]
UniProt Code:	P24385
NCBI GenInfo Identifier:	116152
NCBI Gene ID:	595
NCBI Accession:	P24385.1
UniProt Secondary Accession:	P24385,Q6LEF0,
UniProt Related Accession:	P24385
Molecular Weight:	34kDa
NCBI Full Name:	G1/S-specific cyclin-D1
NCBI Synonym Full Names:	cyclin D1
NCBI Official Symbol:	CCND1
NCBI Official Synonym Symbols:	BCL1; PRAD1; U21B31; D11S287E
NCBI Protein Information:	G1/S-specific cyclin-D1
UniProt Protein Name:	G1/S-specific cyclin-D1
UniProt Synonym Protein Names:	B-cell lymphoma 1 protein; BCL-1; BCL-1 oncogene; PRAD1 oncogene
Protein Family:	G1/S-specific cyclin
UniProt Gene Name:	CCND1

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-Cyclin D1 (Phospho-Thr286) Antibody, Anti-Cyclin D1 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.)