Cyclin D3 (Phospho-Thr283)Colorimetric Cell-Based ELISA Kit (CBCAB00408)

Product Name:	Cyclin D3 (Phospho-Thr283)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00408
ELISA Type:	Cell-Based
Target:	Cyclin D3 (Phospho-Thr283)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Cyclin D3 (Phospho-Thr283) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Cyclin D3 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Cyclin D3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Cyclin D3 phosphorylation.

Qualitative determination of Cyclin D3 (Phospho-Thr283) concentration is achieved by an indirect ELISA format. In essence, Cyclin D3 (Phospho-Thr283) is captured by Cyclin D3 (Phospho-Thr283)-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: 896, UniProt ID: P30281, OMIM: 123834, Unigene: Hs.534307
Gene Symbol:	CCND3
Sub Type:	Phospho

UniProt Protein Function:	CCND3: Regulatory component of the cyclin D3-CDK4 (DC) complex that phosphorylates and inhibits members of the retinoblastoma (RB) protein family including RB1 and regulates the cell-cycle during G(1)/S transition. Phosphorylation of RB1 allows dissociation of the transcription factor E2F from the RB/E2F complex and the subsequent transcription of E2F target genes which are responsible for the progression through the G(1) phase. Hypophosphorylates RB1 in early G(1) phase. Cyclin D-CDK4 complexes are major integrators of various mitogenenic and antimitogenic signals. Also substrate for SMAD3, phosphorylating SMAD3 in a cell-cycle-dependent manner and repressing its transcriptional activity. Component of the ternary complex, cyclin D3/CDK4/p27Kip1, required for nuclear translocation and activity of the cyclin D-CDK4 complex. Interacts with the CDK4 and CDK6 protein kinases to form a serine/threonine kinase holoenzyme complex. The cyclin subunit imparts substrate specificity to the complex. Interacts with ATF5. Interacts with EIF3K. Component of the ternary complex cyclin D/CDK4/p27Kip1 required for nuclear translocation and modulation of CDK4-mediated kinase activity. Can form similar complexes with either p21Cip1 or CDKN2A. Belongs to the cyclin family. Cyclin D subfamily. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Nuclear receptor co-regulator; Cell cycle regulation; Activator Chromosomal Location of Human Ortholog: 6p21 Cellular Component: cyclin-dependent protein kinase holoenzyme complex; cytoplasm; focal adhesion; nucleoplasm; plasma membrane Molecular Function:protein binding; protein kinase binding Biological Process: positive regulation of cyclin-dependent protein kinase activity; positive regulation of protein amino acid phosphorylation
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 through 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 activtiy is required for cell cycle G1/S transition. This protein has been shown to interact with and be involved in the phosphorylation of tumor suppressor protein Rb. The CDK4 activity associated with this cyclin was reported to be necessary for cell cycle progression through G2 phase into mitosis after UV radiation. Several transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Oct 2008]
UniProt Code:	P30281
NCBI GenInfo Identifier:	20981685
NCBI Gene ID:	896
NCBI Accession:	P30281.2
UniProt Secondary Accession:	P30281,Q5T8J0, Q6FG62, Q96F49, B2RD63, B3KQ22, E9PAS4 E9PB36,
UniProt Related Accession:	P30281
Molecular Weight:	9,736 Da
NCBI Full Name:	G1/S-specific cyclin-D3
NCBI Synonym Full Names:	cyclin D3
NCBI Official Symbol:	CCND3
NCBI Protein Information:	G1/S-specific cyclin-D3
UniProt Protein Name:	G1/S-specific cyclin-D3
Protein Family:	G1/S-specific cyclin
UniProt Gene Name:	CCND3
UniProt Entry Name:	CCND3_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-Cyclin D3 (Phospho-Thr283) Antibody, Anti-Cyclin D3 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.)