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.

Products used
LEADseeker Multimodality Imaging System 18-1140-71

Wheat Germ Agglutinin (WGA)-PEI Type A RPNQ0287
PS S PA 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
0.2% BSA (w/v)

GraphPad Prism™ software v4.0 (GraphPad Software)

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.

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.

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


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:11/25/2015)... and HOLLISTON, Mass. , ... Inc. (Nasdaq: HART ), a biotechnology company developing ... CEO Jim McGorry will present at the ... 1, 2015 at 2:30 p.m. PT. The presentation will ... for 30 days. Management will also be available at ...
(Date:11/25/2015)... The Global Genomics Industry ... and in-depth study on the current state of ... ) , The report provides ... classifications, applications and industry chain structure. The Genomics ... including development trends, competitive landscape analysis, and key ...
(Date:11/24/2015)... Halozyme Therapeutics, Inc. (NASDAQ: HALO ) will be presenting at ... on Wednesday, December 2 at 9:30 a.m. ET/6:30 a.m. PT ... provide a corporate overview. th Annual Oppenheimer Healthcare ... ET/10:00 a.m. PT . Jim Mazzola , vice president ... --> th Annual Oppenheimer Healthcare Conference in ...
(Date:11/24/2015)... , Nov. 24, 2015  Clintrax Global, Inc., a worldwide ... Carolina , today announced that the company has set a ... a 391% quarter on quarter growth posted for Q3 of 2014 ... and Mexico , with the establishment of ... December 2015. --> United Kingdom and ...
Breaking Biology Technology:
(Date:11/11/2015)... MINNETONKA, Minn. , Nov. 11, 2015   MedNet ... the entire spectrum of clinical research, is pleased to announce ... Partnerships in Clinical Trials (PCT) event, to be held ... will be able to view live demonstrations of ... platform, and learn how iMedNet has been able ...
(Date:11/9/2015)... 09, 2015 ... the "Global Law Enforcement Biometrics Market ... --> ) has announced the ... Biometrics Market 2015-2019" report to their ... ( ) has announced the addition ...
(Date:11/2/2015)... 2, 2015  SRI International has been awarded a ... development services to the National Cancer Institute (NCI) PREVENT ... scientific expertise, modern testing and support facilities, and analytical ... and toxicology studies to evaluate potential cancer prevention drugs. ... PREVENT Cancer Drug Development Program is an NCI-supported pipeline ...
Breaking Biology News(10 mins):