Navigation Links
Development of an NK2 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System

Key words: NK2 • GPCR receptor binding assay LEADseeker SPA Imaging Beads


Neurokinins, also known as tackykinins, are a family of small neuropeptides. Members of this family include peptides, substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and the recently discovered hemokinin-1 (1). Neurokinins are widely distributed throughout the central and peripheral nervous system where they act via G protein-coupled receptors (NK1, NK2, and NK3 receptors) as neurotransmitters and neuromodulators. They are also considered key mediators in the communication between neurons (particularly sensory neurons) and effector cells (e.g., smooth muscle, glands, and immune cells), a role that is significant to the pathophysiology of a wide variety of diseases including gastrointestinal disorders, emesis, chronic pain, depression, and asthma (2). Members of the neurokinin family favor specific receptors. SP, for instance, shows greatest affinity for NK1 receptors, NKA for NK2, and NKB for NK3 (3). Of these receptors, NK2 receptors are thought to be expressed mainly on smooth muscle cells (4). Additionally, NKA and its truncated form, NKA (4-10), are potent contractors of human colon circular muscle, an action mediated exclusively via NK2 receptors (5).

This application note describes a 384-well NK2 neuropeptide receptor binding assay developed using LEADseeker™ Multimodality Imaging System. The miniaturized assay is robust, achieving a Z’ value of 0.62 and a stability window of 18 h. These factors make the assay suitable for adaptation to automated screening formats.


Materials
Products used
LEADseeker Multimodality Imaging System 18-1140-71

Wheat Germ Agglutinin (WGA)-PEI Type A RPNQ0287
PS SPA Imaging Beads

(2-[125I]iodohistidyl1) Neurokinin A ([125I]Substance K) IM168


Other materials required
Human recombinant NK2 receptor membrane preparation (Euroscreen)

[β-Ala8]-Neurokinin A fragment (4–10) (Sigma)

Costar™ solid white 384-well microplate (Corning)

Protease-free BSA (Sigma)

Buffer: 50 mM Tris pH 7.4
5 mM MgCl2
2 mM EDTA
0.2% BSA (w/v)

GraphPad Prism™ software v4.0 (GraphPad Software)


Protocol
Human recombinant NK2 receptor membrane preparation was used in conjunction with [125I]NKA ligand and WGA-PEI Type A PS SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 5 µM NKA (4–10). The standard assay format was as follows:

1) Reagents were added in the following order: buffer, unlabeled ligand (NSB wells), labeled ligand, membrane, and bead. Total assay volume was 50 µl.

2) Each well contained 10 µl of 0.85 nM [125I]NKA (final concentration 0.17 nM) unless otherwise stated, 2.5 µg of membrane, and 250 µg of beads. Membrane and beads were added in 10 µl volumes.

3) NSB wells contained 20 µl of 13 µM NKA (4–10) (final concentration 5 µM) in addition to the above.

4) Plates were sealed and incubated in darkness for 5 h at room temperature (20–25 °C).

5) Following incubation, plates were imaged on LEADseeker Multimodality Imaging System for 5 min using quasi-coincident averaging and 3 ×3 binning.


Results
Kd determination
Saturation binding was performed with dilutions of [125I]NKA to give a range of concentrations from 0.01 to 1.34 nM in the wells. Figure 1 shows the saturation curve, which was fitted using non-linear regression with the data analysis software package GraphPad Prism v4.0. A Kd value of 0.4 nM (95% confidence intervals 0.3–0.4 nM) was estimated directly from the curve.

IC50 determination
Competitive binding of 0.17 nM [125I]NKA with unlabeled NKA (4–10) was assessed and the IC50 calculated as shown in Figure 2. Final concentrations in the well were 0.002–10 000 nM. Over two experiments, the mean NKA (4–10) IC50 value was determined to be 18.9 nM (95% confidence interval range 13.7–26.1 nM) and mean Ki value was 13.3 nM (95% confidence intervals 9.6–18.3 nM).

Time course
In addition, a time course was performed using standard reagent concentrations detailed in the protocol with an increased incubation time of 18 h. The assay appeared to be stable for the full 18 h as shown in Figure 3.

Z’ analysis
Finally, a Z’ analysis was performed using 42 replicate values for "total" and NSB wells (6). The Z’ value indicated by Figure 4 is 0.62, which was well within the acceptable Z’ value range (0.5–1.0) and confirmed the robustness of the assay.


References
1. Patacchini, R. et al. Trends in Pharmacological Sciences 25, 1–3 (2004).

2. Longmore, J. et al. Canadian Journal Physiology and Pharmacology 75, 612–621 (1997).

3. Regoli, D. et al. Pharmacological Review 46, 551–599 (1994).

4. Lecci, A. et al. British Journal Pharmacology 141, 1249–1263 (2004).

5. Giuliani, S. et al. European Journal Pharmacology 203, 365–370 (1991).

6. Zhang, J. et al. Journal Biomolecular Screening 4 (2), 67–73 (1999).



back to top
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. The let-7 microRNA Family Regulates RAS: Implications for Development and Oncogenesis
2. Rapid Method Development With the BioLogic DuoFlow Chromatography System for the Purification of His-Tagged Proteins
3. A Multiple Mutation Model System as a Test Development and Training Tool for Denaturing Gradient Gel Electrophoresis
4. Development of a Multiplex Bead-Based Assay for Antibody Screening of a Nonhuman Primate Colony on the Bio-Plex System
5. Development of a histamine H1 receptor binding assay using the LEADseeker Multimodality Imaging System
6. Development of a sensitive enzyme fragment complementation assay for cyclic adenosine 3, 5 monophosphate and its validation for pharmacological screening at G protein-coupled receptors
7. Development of an adrenergic α2a receptor binding assay using the LEADseeker Multimodality Imaging System
8. Development of a CRF1 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System
9. Development of Radioligand Binding Assays for the Motilin Receptor Using ScreenReady Targets.
10. The development of a CXCR2 chemokine receptorbinding assay using the LEADseeker Multimodality Imaging System
11. The development of a melanocortin MC4 receptor binding assay using the LEADseeker Multimodality Imaging System
Post Your Comments:
(Date:7/26/2014)... July 26, 2014 Canada Endoscopy ... market data on the Canada Endoscopy Visualization Systems ... US dollars, volume (in units) and average prices ... Endoscopy Visualization Systems, Mid-Range Endoscopy Visualization Systems and ... at http://www.marketresearchreports.biz/analysis/213254 . , The report also ...
(Date:7/25/2014)... July 26, 2014 "Ischemic Stroke ... 2014" provides data on the Ischemic Stroke and ... elemental information and data relating to the clinical ... View full global Ischemic Stroke Research Report 2014 ... overview of the trial numbers and their recruitment ...
(Date:7/25/2014)... 25, 2014 Amgen (NASDAQ: AMGN ... a $0.61 per share dividend for the third quarter ... 5, 2014, to all stockholders of record as of ... About Amgen Amgen is committed to unlocking ... illnesses by discovering, developing, manufacturing and delivering innovative human ...
(Date:7/25/2014)... MA -- Trillions of bacteria live in each person,s ... help digest food and stave off harmful infections, but ... , To help shed light on the role of ... associate professor Eric Alm recently tracked fluctuations in the ... year. The findings, described in the July 25 issue ...
Breaking Biology Technology:Canada Endoscopy Visualization Systems Market Outlook to 2020: MarketResearchReports.Biz 2Canada Endoscopy Visualization Systems Market Outlook to 2020: MarketResearchReports.Biz 3Canada Endoscopy Visualization Systems Market Outlook to 2020: MarketResearchReports.Biz 4Canada Endoscopy Visualization Systems Market Outlook to 2020: MarketResearchReports.Biz 5Ischemic Stroke and Cerebral Stroke Global Clinical Trials Review H2, 2014: MarketResearchReports.Biz 2Ischemic Stroke and Cerebral Stroke Global Clinical Trials Review H2, 2014: MarketResearchReports.Biz 3Amgen Announces 2014 Third Quarter Dividend 2Amgen Announces 2014 Third Quarter Dividend 3Monitoring the rise and fall of the microbiome 2Monitoring the rise and fall of the microbiome 3
... , Evelyn McGown, Ph.D., Jinfang Liao, M.D., Ph.D., Molecular ... Olivier Dery, BD Biosciences Clontech, Palo Alto, CA. ... INTRODUCTION , Proteins inside eukaryotic ... balance between synthesis and degradation. Whereas protein synthesis is ...
... A. Gagne, E. Robitaille, M. Harvey, J.-A. St. Pierre, D. ... Introduction , High throughput ... process is typically highly automated and can provide screening rates over 100,000 ... simple, preferably mix and read type assays, where all reagents necessary to ...
... , This study demonstrated ... platform for a helicase functional assay. Helicase activity was measured on ... the oligos was biotinylated; the other was 5' end-labeled with [ ... a [ 33 P]-biotinylated DNA duplex substrate, then immobilized on the ...
Cached Biology Technology:Measurement of proteasome inhibition in live cells in Molecular Devices microplate fluorometers 2Measurement of proteasome inhibition in live cells in Molecular Devices microplate fluorometers 3Measurement of proteasome inhibition in live cells in Molecular Devices microplate fluorometers 4Measurement of proteasome inhibition in live cells in Molecular Devices microplate fluorometers 5Receptor Binding Assays using Fluorescence Polarization Detection: the [FP]2TM Characterized Tracers 2Receptor Binding Assays using Fluorescence Polarization Detection: the [FP]2TM Characterized Tracers 3Receptor Binding Assays using Fluorescence Polarization Detection: the [FP]2TM Characterized Tracers 4Receptor Binding Assays using Fluorescence Polarization Detection: the [FP]2TM Characterized Tracers 5Receptor Binding Assays using Fluorescence Polarization Detection: the [FP]2TM Characterized Tracers 6Evaluation of FlashPlate in a Helicase Assay 2Evaluation of FlashPlate in a Helicase Assay 3
(Date:7/25/2014)... of Adelaide has opened the way for the development of ... In Australia, annual barley production is second only to wheat ... of the most important diseases of barley. , Senior Research ... of special growths on the cell walls of barley plants ... , The research, by the ARC Centre of Excellence in ...
(Date:7/25/2014)... (SB) is a complex congenital central nervous system ... of the neural tubes during the embryonic phase. ... and fecal incontinence and neurocognitive retardation. Such problems ... of life. Researchers at Ankara Physical Medicine and ... functional performance in children with SB, using the ...
(Date:7/25/2014)... reach puberty is influenced by ,imprinted genes,a subset of genes ... This is the first evidence that imprinted genes can ... this study were published today in the journal Nature ... is a marker for the timing of puberty in females. ... between girls, is an inherited trait, and is linked to ...
Breaking Biology News(10 mins):New hope for powdery mildew resistant barley 2Could age of first period influence development of diseases in older women? 2
... the International Symposium " Nature and Governance Biodiversity Data, Science, ... 11 to 12 February. The symposium aimed at clarifying and popularizing ... Meeting held from 13 to 15 February 2013. The ... and brought together high-ranking speakers and guests from across the world ...
... chair!" That command given by countless parents to their ... a robot that is actually a computerized model of a ... a University of Iowa research program. That,s because a ... to accurately predict the role posture plays in transferring the ...
... A team of UCLA-led researchers has identified a protein ... as a weapon against deadly human pathogenic viruses such ... designated "priority pathogens" for national biosecurity purposes by the ... In a study published in the January issue ...
Cached Biology News:Virtual vehicle vibrations 2Newly identified natural protein blocks HIV, other deadly viruses 2
Mouse monoclonal antibody raised against a partial recombinant AAAS. NCBI Entrez Gene ID = AAAS...
... Cosmid Cloning Kit enables you to prepare, ... unbiased primary cosmid library of genomic DNA ... sublibraries. High efficiency cosmid library construction is ... protocols. First, genomic DNA is randomly sheared ...
Mouse monoclonal antibody raised against a partial recombinant EBI3. NCBI Entrez Gene ID = EBI3...
... VersaDoc MP 4000 System is a ... system capable of supporting a wide ... chemiluminescence, densitometry, and chemiflourescence. Red, ... imaging of multiplexed fluorescent samples, reducing ...
Biology Products: