Rat DCN (Decorin) ELISA Kit (RTFI00727)- High Sensitivity

Product Name:	Rat DCN (Decorin) ELISA Kit
Product Code:	RTFI00727
Size:	96 Assays
Target:	Rat DCN
Alias:	DCN, DSPG2, PG-II, PGS2, SLRR1B, Bone proteoglycan II, CSCD, decorin, decorin proteoglycan, dermatan sulphate proteoglycans II, DSPG2, PG40, PGII, PG-S2, proteoglycan core protein, SLRR1BPGS2, small leucine-rich protein 1B
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	4.688pg/ml
Range:	7.813-500pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

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

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-102	95
EDTA plasma(n=5)	86-104	92
UFH plasma(n=5)	92-103	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat DCN 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)	92-105%	87-105%	89-104%
EDTA plasma(n=5)	82-100%	83-99%	83-99%
UFH plasma(n=5)	83-97%	81-97%	82-99%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

Q01129

UniProt Protein Function:	DCN: May affect the rate of fibrils formation. Defects in DCN are the cause of congenital stromal corneal dystrophy (CSCD). Corneal dystrophies are inherited, bilateral, primary alterations of the cornea that are not associated with prior inflammation or secondary to systemic disease. Most show autosomal dominant inheritance. Belongs to the small leucine-rich proteoglycan (SLRP) family. SLRP class I subfamily. 5 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Motility/polarity/chemotaxis; Extracellular matrix; Secreted Cellular Component: extracellular matrix; extracellular space; proteinaceous extracellular matrix; collagen type VI Molecular Function:collagen binding; glycosaminoglycan binding; extracellular matrix binding; protein N-terminus binding Biological Process: skeletal muscle development; wound healing; response to mechanical stimulus; response to lipopolysaccharide; kidney development; peptide cross-linking via chondroitin 4-sulfate glycosaminoglycan; placenta development; aging
NCBI Summary:	binds the N-terminal region of collagen VI; may play a role in extracellular matrix assembly [RGD, Feb 2006]
UniProt Code:	Q01129
NCBI GenInfo Identifier:	54020664
NCBI Gene ID:	29139
NCBI Accession:	NP_077043.1
UniProt Related Accession:	Q01129
Molecular Weight:	39,805 Da
NCBI Full Name:	decorin
NCBI Synonym Full Names:	decorin
NCBI Official Symbol:	Dcn
NCBI Protein Information:	decorin
UniProt Protein Name:	Decorin
UniProt Synonym Protein Names:	Bone proteoglycan II; Dermatan sulfate proteoglycan-II; DSPG; PG-S2; PG40
Protein Family:	Decorin
UniProt Gene Name:	Dcn
UniProt Entry Name:	PGS2_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.