Rat Cyclin D3/CCND3 ELISA Kit (RTFI00641)

Product Name:	Rat Cyclin D3/CCND3 ELISA Kit
Product Code:	RTFI00641
Size:	96 Assays
Target:	Rat CCND3
Alias:	CCND3, CCND3, cyclin D3, D3-type cyclin, G1, S-specific cyclin D3, G1, S-specific cyclin-D3
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	18.75pg/ml
Range:	31.25-2000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat CCND3 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat CCND3 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-101	92
EDTA plasma(n=5)	86-103	94
UFH plasma(n=5)	87-101	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat CCND3 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	86-105%	97-105%	85-95%
EDTA plasma(n=5)	85-101%	91-101%	85-96%
UFH plasma(n=5)	80-98%	83-96%	82-100%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P48961

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; Activator; Cell cycle regulation Cellular Component: cyclin-dependent protein kinase holoenzyme complex; cytoplasm; focal adhesion; nucleus; plasma membrane Molecular Function:cyclin-dependent protein kinase activity; protein kinase binding Biological Process: G1/S transition of mitotic cell cycle; hyaluronan biosynthetic process; positive regulation of cell proliferation; positive regulation of cyclin-dependent protein kinase activity; positive regulation of protein amino acid phosphorylation; regulation of cell cycle; response to glucose stimulus; response to organic substance; response to peptide hormone stimulus; signal transduction; T cell proliferation
NCBI Summary:	cyclin that binds both Cdk4 and Cdk6; involved in cell cycle regulation [RGD, Feb 2006]
UniProt Code:	P48961
NCBI GenInfo Identifier:	1345742
NCBI Gene ID:	25193
NCBI Accession:	P48961.1
UniProt Secondary Accession:	P48961,Q63628,
UniProt Related Accession:	P48961
Molecular Weight:	32,434 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_RAT

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37°C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum:	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum and assay promptly or aliquot and store the samples at-80°C. Avoid multiple freeze-thaw cycles.
Plasma:	Collect plasma using EDTA or heparin as an anti-coagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid:	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell Culture Supernatant:	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell Lysates:	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.
Tissue Homogenates:	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenizein 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or-80°C.
Tissue Lysates:	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk:	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.