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NA / NE / Noradrenaline / Norepinephrine ELISA Kit

SKU:
UNFI0040
Product Type:
ELISA Kit
Size:
96 Assays
Sensitivity:
9.375pg/ml
Range:
15.625-1000pg/ml
ELISA Type:
Competitive
Synonyms:
NA, NE, Noradrenaline, Norepinephrine
Reactivity:
Universal
€649
Frequently bought together:

Description

NA / NE / Noradrenaline / Norepinephrine ELISA Kit

Noradrenaline/Norepinephrine is a neurotransmitter and hormone involved in various physiological processes in the body. Its functions include regulating the "fight or flight" response, attention, mood, and the autonomic nervous system. The Assay Genie noradrenaline ELISA kit can be used to measure and quantify levels of noradrenaline in biological samples, aiding in the study of its role in different conditions and diseases.

system_update_alt Datasheet system_update_alt MSDS

Key Features

Save Time Pre-coated 96 well plate
Quick Start Kit includes all necessary reagents
Publication Ready Reproducible and reliable results

Overview

Product Name:

NA/NE (Noradrenaline/Norepinephrine) ELISA Kit

Product Code:

UNFI0040

Size:

96 Assays

Alias:

NA, NE, Noradrenaline, Norepinephrine

Detection Method:

Competitive ELISA, Coated with Antibody

Reactivity::

Universal

Sensitivity:

9.375pg/ml

Range:

15.625-1000pg/ml

Storage:

4°C for 6 months

Note:

For Research Use Only

Additional Information

Recovery

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

Matrix

Recovery Range (%)

Average (%)

serum (n=5)

90-101

96

EDTA plasma (n=5)

85-104

95

UFH plasma (n=5)

85-102

91

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of NA/NE 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)

87-105%

86-100%

85-102%

EDTA plasma (n=5)

88-98%

87-100%

87-101%

UFH Plasma (n=5)

83-97%

80-97%

80-98%

CV(%)

Intra Assay <8

Inter Assay <10

Kit Components

Component Quantity Storage

ELISA Microplate (Dismountable)

8x12 strips

2-8°C/-20°C

Lyophilized Standard

2

2-8°C/-20°C

Sample/Standard Dlution Buffer

20ml

2-8°C

Biotin-labeled Antibody (Concentrated)

120ul

2-8°C (Protection from light)

Antibody Dilution Buffer

10ml

2-8°C

HRP-Streptavidin Conjugate (SABC)

120ul

2-8°C (Protect from light)

SABC Dilution Buffer

10ml

2-8°C

TMB Substrate

10ml

2-8°C (Protection from light)

Stop Solution

10ml

2-8°C

Wash Buffer (25X)

30ml

2-8°C

Plate Sealer

5

-

Other materials 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

Protocol

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

Equilibrate the TMB substrate for at least 30 min at 37°C beforeuse. When diluting samples and reagents, they must be mixed completely andevenly. It is recommended to plot a standard curve for each test.

Step Procedure

1.

Set standard, test sample and control (zero) wells on the pre-coatedplate respectively, and then, record their positions. It isrecommended to measure each standard and sample in duplicate. Washplate 2 times before adding standard, sample and control (zero) wells!

2.

Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank or Sample per well. The blankwell is added with Sample Dilution Buffer. Immediately add 50 µL of biotin-labelled antibody workingsolution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thoroughmixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA platewell, avoid touching plate walls and foaming).

3.

Wash: Aspirate each well and wash, repeating the process three timesWash by filling each well with Wash Buffer (approximately 350µL)using a squirt bottle, multi-channel pipette, manifold dispenser orautomated washer. Complete removal of liquid at each step is essentialto good performance. After the last wash, remove any remaining WashBuffer by aspirating or decanting. Invert the plate and pat it againstthick clean absorbent paper.

4.

HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC workingsolution to each well. Cover with a new Plate sealer. Incubate for30minutes at 37°C.

5.

Wash: Repeat the aspiration/wash process for five times.

6.

TMB Substrate: Add 90µL of TMB Substrate to each well. Coverwith a new Plate sealer. Incubate for about 10-20 minutes at 37°C.Protect from light. The reaction time can be shortened or extendedaccording to the actual color change, but not more than 30minutes.When apparent gradient appeared in standard wells, you can terminatethe reaction.

7.

Stop: Add 50µL of Stop Solution to each well. Color turn toyellow immediately. The adding order of stop solution should be as thesame as the substrate solution.

8.

OD Measurement: Determine the optical density (OD Value) of each wellat once, using a microplate reader set to 450 nm. You should open themicroplate reader ahead, preheat the instrument, and set the testing parameters.

Sample Preparation

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.

Noradrenaline Background

What is noradrenaline/norepinephrine?

Norepinephrine, also known as noradrenaline, is a neurotransmitter and hormone that plays a vital role in the functioning of the human body. It is a member of the catecholamine family, which also includes dopamine and epinephrine. Norepinephrine is synthesized and released by neurons in specific regions of the brain, as well as by the adrenal glands, where it functions as a hormone. It is involved in regulating various physiological processes, including the "fight or flight" response, attention, mood, and the autonomic nervous system.

Norepinephrine Structure

Structurally, norepinephrine is derived from the amino acid tyrosine and consists of a benzene ring with two hydroxyl groups (-OH) and an amine group (-NH2). It closely resembles dopamine, differing by the addition of a methyl group (-CH3) to the amino group. The presence of both hydroxyl groups makes norepinephrine more polar than dopamine, enabling it to be stored and transported in vesicles within nerve terminals until its release is triggered by neuronal activity.

Structure of Norepinephrine. Source: PubChem

Norepinephrine Synthesis

The synthesis of norepinephrine involves a series of enzymatic reactions within the human body. It begins with the conversion of the amino acid tyrosine into L-DOPA by the enzyme tyrosine hydroxylase. L-DOPA is subsequently decarboxylated to dopamine by the enzyme aromatic L-amino acid decarboxylase. From there, dopamine undergoes hydroxylation by dopamine β-hydroxylase, resulting in the synthesis of norepinephrine.

This enzymatic cascade primarily occurs within neurons located in specific regions of the brain, including the locus coeruleus and the sympathetic ganglia. Additionally, norepinephrine synthesis takes place in the adrenal medulla, where it serves as a precursor for the synthesis of epinephrine. This tightly regulated synthesis pathway ensures the production of adequate norepinephrine levels for its vital physiological functions throughout the body.

Functions of Norepinephrine

Norepinephrine serves as a principal modulator of the sympathetic nervous system, orchestrating the physiological responses associated with the "fight or flight" reaction. By stimulating the release of norepinephrine, the body initiates a series of adaptive changes, including heightened heart rate, augmented blood pressure, and enhanced blood flow to the muscles, effectively preparing the organism for action. Additionally, norepinephrine contributes to attention regulation, arousal modulation, and mood control, with implications for conditions such as depression, anxiety disorders, and attentional deficits.

Norepinephrine Receptors

The actions of norepinephrine are mediated through adrenergic receptors that are ubiquitously distributed across various tissues. These receptors encompass two primary categories: α (alpha) and β (beta), further divided into subtypes. α receptors exhibit α1 and α2 subcategories, while β receptors comprise β1, β2, and β3 subtypes. Each receptor subtype governs distinct physiological effects. For instance, α1 receptor activation elicits vasoconstriction, whereas α2 receptor stimulation can lead to autoregulatory inhibition of norepinephrine release. β1 receptors predominate in the cardiac tissue and regulate heart rate and contractility, while β2 receptors predominantly exist in smooth muscle tissues and mediate bronchodilation and vasodilation.

Norepinephrine Mechanism of Action

The mechanism of action of norepinephrine entails its binding to adrenergic receptors on target cells. Upon release from presynaptic nerve terminals, norepinephrine diffuses into the synaptic cleft and interacts with these receptors, initiating intracellular signaling cascades. Adrenergic receptors are coupled with G-proteins, which facilitate the activation of various effector systems and second messenger pathways. For instance, α1 receptor activation induces calcium ion release from intracellular stores, thereby fostering smooth muscle contraction. In contrast, β receptor activation stimulates the production of cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger that modulates numerous cellular processes. Through these intricate mechanisms, norepinephrine exerts its pleiotropic effects on target tissues and organs, thereby ensuring homeostasis maintenance and facilitating adaptive responses to physiological demands.

Norepinephrine Disorders

Alterations in norepinephrine signaling have been associated with various disorders. An imbalance in norepinephrine levels is implicated in psychiatric conditions like depression, anxiety, and attention-deficit/hyperactivity disorder (ADHD). Drugs that modulate norepinephrine activity, such as selective norepinephrine reuptake inhibitors (SNRIs), are commonly used in the treatment of depression and certain anxiety disorders. In addition, dysregulation of norepinephrine signaling is linked to cardiovascular diseases, including hypertension and congestive heart failure. Understanding the role of norepinephrine and its associated disorders is crucial for the development of targeted therapies aimed at restoring normal physiological function and alleviating symptoms in affected individuals.

NA/NE ELISA Kits FAQs

Q: What is the purpose of a norepinephrine ELISA kit?

The norepinephrine ELISA kit is designed for the quantitative measurement of norepinephrine levels in biological samples, aiding in the study of its role in various physiological processes and associated disorders.

Q: What sample types can be used with the norepinephrine ELISA kit?

The norepinephrine ELISA kit can be used with a range of sample types, including plasma, serum, urine, and tissue extracts.

Q: Is the norepinephrine ELISA kit suitable for cross-species studies?

Yes, the norepinephrine ELISA kit is designed for universal reactivity, allowing analysis of norepinephrine from different species, including humans, rodents, primates, and others.

Q: Where can I find additional technical support or assistance with the norepinephrine ELISA kit?

For any technical inquiries or assistance regarding the norepinephrine ELISA kit, you can reach out to our team. They will be available to answer your questions and provide the necessary guidance to ensure a successful experiment.

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