Rat Neuropeptide Y/NPY ELISA Kit (RTFI00090)

Product Name:	Rat Neuropeptide Y/NPY ELISA Kit
Product Code:	RTFI00090
Size:	96 Assays
Target:	Rat Npy
Alias:	Npy, Neuropeptide Y
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 Npy and the recovery rates were calculated by comparing the measured value to the expected amount of Rat Npy in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	93-100	97
EDTA plasma(n=5)	87-105	96
UFH plasma(n=5)	87-100	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat Npy 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)	92-103%	89-104%	91-105%
EDTA plasma(n=5)	91-100%	91-101%	84-101%
UFH plasma(n=5)	81-97%	84-93%	90-100%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P07808

UniProt Protein Function:	NPY: NPY is implicated in the control of feeding and in secretion of gonadotrophin-release hormone. Belongs to the NPY family.
UniProt Protein Details:	Protein type:Hormone; Secreted; Secreted, signal peptide Cellular Component: Golgi apparatus; extracellular space; cytoplasm Molecular Function:neuropeptide hormone activity; G-protein-coupled receptor binding; neuropeptide Y receptor binding; neuropeptide Y receptor activity Biological Process: synaptic transmission; sensory perception of taste; neuropeptide signaling pathway; positive regulation of appetite; regulation of blood pressure; adult feeding behavior; feeding behavior; cerebral cortex development; negative regulation of blood pressure; neurite development; central nervous system neuron development
NCBI Summary:	This gene encodes a neuropeptide that is widely expressed in the central nervous system and influences many physiological processes, including cortical excitability, stress response, food intake, circadian rhythms, and cardiovascular function. Studies in the rat model of depression (Flinders Sensitive Line) show that this gene is downregulated in the hippocampus and the prefrontal cortex compared to the control ((Flinders Resistant Line). Alternatively spliced transcript variants of this gene have been described, but the function of all the variants is not known. [provided by RefSeq, Jul 2012]
UniProt Code:	P07808
NCBI GenInfo Identifier:	6981286
NCBI Gene ID:	24604
NCBI Accession:	NP_036746.1
UniProt Related Accession:	P07808
Molecular Weight:	Predicted Molecular Mass: 37.8kDaAccurate Molecular Mass: 38kDa as determined by SDS-PAGE reducing conditions.
NCBI Full Name:	pro-neuropeptide Y preproprotein
NCBI Synonym Full Names:	neuropeptide Y
NCBI Official Symbol:	Npy
NCBI Official Synonym Symbols:	NPY02; RATNPY; RATNPY02
NCBI Protein Information:	pro-neuropeptide Y
UniProt Protein Name:	Pro-neuropeptide Y
UniProt Synonym Protein Names:	Neuropeptide tyrosine
Protein Family:	Neuropeptide
UniProt Gene Name:	Npy
UniProt Entry Name:	NPY_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.