Product Name:	Rat G1/S-specific cyclin-D1 (Ccnd1) ELISA Kit
SKU:	RTEB0356
Size:	96T
Target:	Rat G1/S-specific cyclin-D1 (Ccnd1)
Synonyms:	G1/S-specific cyclin-D1, Ccnd1
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	0.312-20ng/mL
Sensitivity:	0.163ng/mL

Intra CV:

4.8%

Inter CV:

8.3%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	105-115%	96-106%	81-94%	88-99%
EDTA Plasma(N=5)	111-119%	103-112%	110-120%	94-103%
Heparin Plasma(N=5)	85-94%	90-100%	99-109%	97-106%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	93	87-99
Plasma	95	89-101

Function:

Regulatory component of the cyclin D1-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 D1/CDK4/CDKN1B, required for nuclear translocation and activity of the cyclin D-CDK4 complex, Exhibits transcriptional corepressor activity with INSM1 on the NEUROD1 and INS promoters in a cell cycle-independent manner.

Uniprot:	P39948
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat G1/S-specific cyclin-D1
Sub Unit:	Interacts with either CDK4 or CDK6 protein kinase to form a serine/threonine kinase holoenzyme complex. The cyclin subunit imparts substrate specificity to the complex. Component of the ternary complex cyclin D/CDK4/CDKN1B required for nuclear translocation and modulation of CDK4-mediated kinase activity. Interacts directly with CDKN1B. Can form similar complexes with either CDKN1A or CDKN2A. Interacts with USP2 and FBXO4. Interacts with UHRF2; the interaction ubiquitinates CCND1 and appears to occur independently of phosphorylation. Interacts (via cyclin N-terminal domain) with INSM1 (via N-terminal region); the interaction competes with the binding of CCND1 to CDK4 during cell cycle progression and inhibits CDK4 activity. Interacts with CDK4; the interaction is prevented with the binding of CCND1 to INSM1 during cell cycle progression.
Research Area:	Cancer
Subcellular Location:	Nucleus Cytoplasm Membrane Cyclin D-CDK4 complexes accumulate at the nuclear membrane and are then translocated into the nucleus through interaction with KIP/CIP family members.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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:Oncoprotein; Activator; Cell cycle regulation; Nuclear receptor co-regulator Cellular Component: nucleoplasm; tight junction; transcriptional repressor complex; membrane; cytoplasm; cyclin-dependent protein kinase holoenzyme complex; intracellular; nucleus Molecular Function:enzyme binding; histone deacetylase binding; protein complex binding; kinase activity; cyclin-dependent protein kinase regulator activity; transcription corepressor activity; transcription factor binding; protein kinase binding; protein kinase activity Biological Process: G1 DNA damage checkpoint; lactation; fat cell differentiation; re-entry into mitotic cell cycle; negative regulation of Wnt receptor signaling pathway; positive regulation of cyclin-dependent protein kinase activity; regulation of cell cycle; response to glucocorticoid stimulus; positive regulation of mammary gland epithelial cell proliferation; negative regulation of epithelial cell differentiation; Wnt receptor signaling pathway through beta-catenin; negative regulation of transcription from RNA polymerase II promoter; response to organic cyclic substance; protein amino acid phosphorylation; response to estradiol stimulus; response to organic substance; response to magnesium ion; response to vitamin E; Leydig cell differentiation; positive regulation of cell proliferation; response to iron ion; response to corticosterone stimulus; response to X-ray; response to drug; organ regeneration; Wnt receptor signaling pathway; transcription, DNA-dependent; unfolded protein response; mammary gland epithelial cell proliferation; liver development; response to organic nitrogen; response to ethanol; cell division; response to steroid hormone stimulus; response to estrogen stimulus; positive regulation of protein amino acid phosphorylation; response to calcium ion; regulation of protein kinase activity; response to DNA damage stimulus; G1/S transition of mitotic cell cycle
NCBI Summary:	regulatory subunit of CDK4 or CDK6; involved in regulation of the G1/S transition of the cell cycle [RGD, Feb 2006]
UniProt Code:	P39948
NCBI GenInfo Identifier:	148356229
NCBI Gene ID:	58919
NCBI Accession:	NP_741989.3
UniProt Related Accession:	P39948
Molecular Weight:	Predicted Molecular Mass: 34.7kDaAccurate Molecular Mass: 34kDa as determined by SDS-PAGE reducing conditions.
NCBI Full Name:	G1/S-specific cyclin-D1
NCBI Synonym Full Names:	cyclin D1
NCBI Official Symbol:	Ccnd1
NCBI Protein Information:	G1/S-specific cyclin-D1
UniProt Protein Name:	G1/S-specific cyclin-D1
Protein Family:	G1/S-specific cyclin
UniProt Gene Name:	Ccnd1
UniProt Entry Name:	CCND1_RAT

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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 clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum 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 anticoagulant. 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 & homogenize in 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.

Rat G1/S-specific cyclin-D1 (Ccnd1) ELISA Kit (RTEB0356)

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