Mouse Neuropeptide Y ELISA Kit is a sandwich Enzyme-Linked Immunosorbent Assay (sELISA) designed for the in vitro quantitative determination of mouse Neuropeptide Y in serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids.

Assay Type

Sandwich (quantitative)

Principle of Assay

Mouse Neuropeptide Y ELISA Kit (A3330) employs the sandwich enzyme immunoassay technique for the quantitative measurement of mouse Neuropeptide Y in serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids. An antibody specific for Neuropeptide Y has been pre-coated onto a 96-well microtiter plate. The standards and test samples are added into the wells and the Neuropeptide Y present in each sample is bound to the wells by the immobilized antibody. Following incubation, the wells are washed and then incubated with Biotinylated Anti-Neuropeptide Y Antibody, which binds the captured Neuropeptide Y present in each well. Following incubation, unbound biotinylated detection antibody is removed by washing, and an HRP-Avidin conjugate is added to the wells and the microtiter plate is incubated. Following incubation and washing, TMB substrate solution is then used to visualize the HRP enzymatic reaction by catalysis to produce a blue-coloured product that changes to yellow after addition of acidic stop solution. The density of yellow is proportional to the amount of Neuropeptide Y captured in each well. The concentration of Neuropeptide Y can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.

Platform

Pre-coated Microplate (12 x 8 well strips)

Detection Type

Colourmetric

Instrument

Colorimetric Microplate Reader

Sample Type

Serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids.

Sensitivity

4.4 pg/ml

Product Range

15.6-1000 pg/ml

Assay Time

4h 30m

Reactivity

Mouse

Recovery

Sample Type	n	Range	Average
Serum	5	80% - 102%	91%
EDTA Plasma	5	81% - 100%	90%
Heparin Plasma	5	80% - 89%	84%

Linearity

Sample Type	1:2	1:4	1:8	1:16
Serum (n=5)	87-91%	87-107%	74-101%	92-97%
EDTA Plasma (n=5)	90-105%	84-101%	90-101%	79-108%
Heparin Plasma (n=5)	84-95%	92-105%	82-105%	89-91%

Precision

Intra-assay: CV < 10%, Inter-assay: CV < 12%

General Notes

Information online is representative. Always refer to the Product Manual inside the kit.

Storage

Store kit components at +4°C or -20°C as instructed. Do not use past expiration date!

Synonyms

C-flanking peptide of NPY, CPON, Neuropeptide tyrosine, Neuropeptide Y precursor, NPY, NPY_HUMAN, Pro neuropeptide Y, PYY 4, PYY4, Y Neuropeptide

Disclaimer

This product is for research use only. It is not intended for diagnostic or therapeutic use.

Kit Components

Item	Quantity	Storage
Pre-Coated 96 Well Microplate	12 x 8 Well Strips	-20°C
Lyopholized Standard	2 Vials	-20°C
Detection Solution A	120μl	-20°C
Detection Solution B	120µl	-20°C
Wash Buffer (30X)	20ml	+4°C
Sample Dilution Buffer	45ml	-20°C
TMB Substrate	9ml	+4°C
Stop Solution	6ml	+4°C
Plate Sealers	5 Adhesive Strips	-

Required Materials

The following materials are not included in the kit, but are required to run this assay:

Microplate reader capable of measuring absorbance at 450nm ± 10nm.
Squirt bottle, multichannel pipette reservoir, or automated microplate washer.
Graph paper or computer software capable of generating logarithmic functions.
Test tubes and Eppendorf tubes to prepare standards and samples.
Multi- and single-channel pipettes and sterile pipette tips.
Deionized or distilled water.
Absorbent paper towels.
37°C incubator.

Important Notes

Sample concentrations should be estimated before being used in the assay and a proper dilution factor should be selected to make the diluted target protein concentration fall in the optimal detection range of this kit.
Hemolysis can greatly impact the validity of test results. Take care to minimize hemolysis.
Samples should be brought to room temperature before starting the assay.

Bring all reagents and samples to room temperature before use.

We recommend that all standards and samples be assayed in duplicate.

Step 1

Prepare all reagents, samples, and working standards as directed in the previous sections.

Step 2

Determine the number of microplate strips required for the assay and insert them into the plate frame. Unused strips should be stored in a foil pouch at -20°C.

Step 3

Add 100μl of standard solutions to the standard wells. Note: A plate layout is provided to record standards and samples assayed.

Step 4

Add 100μl of samples to the sample wells.

Step 5

Cover with an adhesive plate sealer provided and incubate at 37°C for 2 hours.

Step 6

Remove the plate sealer and aspirate the liquid from the plate.

Step 7

Add 100μl of working Detection Solution A to each well.

Step 8

Cover with a new adhesive plate sealer and incubate at 37°C for 60 minutes.

Step 9

Remove the plate sealer, aspirate the liquid from the plate, and wash the plate three times with Wash Buffer. Soak wells with 300μl of Wash Buffer for 1-2 minutes each time. Note: Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Blot the plate onto paper towels or other absorbent material. Do not let the wells dry completely at any time!

Step 10

Add 100μl of working Detection Solution B to each well.

Step 11

Cover with a new adhesive plate sealer and incubate at 37°C for 60 minutes.

Step 12

Remove the plate sealer, aspirate the liquid from the plate, and wash the plate five times with Wash Buffer. Soak wells with 300μl of Wash Buffer for 1-2 minutes each time.

Step 13

Add 90μl of TMB Substrate to each well. Cover with a new plate sealer and incubate at 37°C in the dark for 15-25 minutes (do not exceed 30 minutes). Note: This incubation time is for reference only, the optimal reaction time should be determined by the end-user and can be shortened or extended according to the actual color change. The reaction may be terminated when a gradient is apparent in the standard wells.

Step 14

Add 50μl of Stop Solution to each well. The color in the wells should change from blue to yellow immediately. Note: If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.

Step 15

Determine the optical density (O.D. value) of each well immediately using a microplate reader set to 450 nm.

Scientific Validation Data (1)

Enlarge Image

Figure 1: Standard Curve - Mouse Neuropeptide Y ELISA Kit (A3330)

Representative standard curve for Mouse Neuropeptide Y ELISA Kit (A3330).

Citations (1)

View Publication

Wide-Ranging Effects on the Brain Proteome in a Transgenic Mouse Model of Alzheimer's Disease Following Treatment with a Brain-Targeting Somatostatin Peptide

References: Mouse Neuropeptide Y ELISA Kit (A3330)

Abstract:

Alzheimer's disease is the most common neurodegenerative disorder characterized by the pathological aggregation of amyloid-ß (Aß) peptide. A potential therapeutic intervention in Alzheimer's disease is to enhance Aß degradation by increasing the activity of Aß-degrading enzymes, including neprilysin. The somatostatin (SST) peptide has been identified as an activator of neprilysin. Recently, we demonstrated the ability of a brain-penetrating SST peptide (SST-scFv8D3) to increase neprilysin activity and membrane-bound Aß42 degradation in the hippocampus of mice overexpressing the Aß-precursor protein with the Swedish mutation (APPswe). Using LC-MS, we further evaluated the anti-Alzheimer's disease effects of SST-scFv8D3. Following a triple intravenous injection of SST-scFv8D3, the LC-MS analysis of the brain proteome revealed that the majority of downregulated proteins consisted of mitochondrial proteins regulating fatty acid oxidation, which are otherwise upregulated in APPswe mice compared to wild-type mice. Moreover, treatment with SST-scFv8D3 significantly increased hippocampal levels of synaptic proteins regulating cell membrane trafficking and neuronal development. Finally, hippocampal concentrations of growth-regulated a (KC/GRO) chemokine and degradation of neuropeptide-Y were elevated after SST-scFv8D3 treatment. In summary, our results demonstrate a multifaceted effect profile in regulating mitochondrial function and neurogenesis following treatment with SST-scFv8D3, further suggesting the development of Alzheimer's disease therapies based on SST peptides.

View Publication

Publishing research using Mouse Neuropeptide Y ELISA Kit (A3330)? Please let us know so that we can list the citation on this page.