Navigation Links
Development of an adrenergic α2a receptor binding assay using the LEADseeker Multimodality Imaging System

Key words: α2c receptor GPCR receptor binding assay LEADseeker SPA Imaging Beads


The α2-adrenergic receptors are G protein-coupled receptors that play a key role in the control of numerous physiological functions such as renal Na+ re-absorption, insulin secretion, platelet aggregation, and neurotransmitter release at sympathetic nerve endings (1). They consist of three highly homologous subtypes encoded by distinct genes: α2a, α2b, and α2c. Both α2a and α2c subtypes are required for normal presynaptic control of neurotransmitter release from sympathetic nerves from the heart and from central noradrenergic neurons. α2a adrenergic receptors inhibit transmitter release at high stimulation frequencies whilst the α2c subtype modulates neurotransmission at lower levels of nerve activity (2). The α2c receptor is distinguishable from α2a and α2b receptors by its sensitivity to prazosin (3).

This application note describes a 384-well adrenergic α2c receptor binding assay developed using the LEADseeker™ Multimodality Imaging System.


Materials
Products used

LEADseeker Multimodality Imaging System 18-1140-71

Wheat Germ Agglutinin (WGA) YOx RPNQ0270
SPA Imaging Beads

[0-methyl-3H]Rauwolscine TRK685


Other materials required
Human recombinant adrenergic α2c receptor membrane preparation (Euroscreen, ES-032-M)

Prazosin hydrochloride (Sigma, P7791)

Rauwolscine hydrochloride (Sigma, R104)

Costar™ solid white 384-well microplate (Corning, 3705)

Buffer: 50-mM Tris pH 7.4
5-mM MgCl2
50-mM NaCl

GraphPad Prism™ software v4.0 (GraphPad Software)


Protocol
Human recombinant α2c receptor membrane preparation was used in conjunction with [0-methyl-3H]Rauwolscine ligand and WGA YOx SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 6-µM prazosin. The standard assay format was as follows:

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

2. Diluted membrane and bead were precoupled for 30 min on a roller mixer at 2–8 ºC.

3. Wells contained 10 µl of 6.5-nM [0-methyl3H]Rauwolscine (final concentration 1.3 nM) unless otherwise stated. Wells also contained 5 µg of membrane and 250 µg of WGA YOx SPA Imaging Beads. Precoupled bead and membrane was added in a 20-µl volume.

4. NSB wells contained 10 µl of 30-µM prazosin (final concentration 6 µM) in addition to the above.

5. Plates were sealed and incubated in darkness for 3 h at room temperature (20–25 ºC) unless otherwise stated.

6. Following incubation, plates were imaged on the 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 [0-methyl-3H]Rauwolscine to give a range of concentrations from 0.07 to 8.8 nM in the wells. Plates were incubated for 5 h prior to imaging. Figure 1 shows the saturation curve, which was fitted using nonlinear regression with the data analysis software package GraphPad Prism v4.0. Over two experiments the mean Kd value was determined to be 1.3 nM (95% confidence intervals 0.8 to 1.9 nM), estimated directly from the curve. Figure 1 is representative of the data obtained.

DMSO tolerance
Dilutions of DMSO were prepared in assay buffer to give a range of concentrations from 0.25% (v/v) to 5% (v/v) in the well. The results shown in Figure 2 indicate the assay was tolerant to DMSO up to a final concentration of 1% (v/v).

IC50 determination
Competitive binding of 1.3-nM [0-methyl-3H]Rauwolscine with rauwolscine (Fig 3A) and prazosin (Fig 3B) was assessed and the IC50 value of each ligand calculated. Final concentrations in the wells were from 0.0004 to 4000 nM and 0.25 to 20000 nM respectively. DMSO was present at a final concentration of 1% v/v in each well.

Over two experiments, the mean rauwolscine IC50 value was determined to be 3.1 nM (95% confidence interval range 2.7–3.7 nM) and the mean Ki value was 1.6 nM (95% confidence intervals 1.3–1.8 nM). The mean prazosin IC50 value was determined to be 430 nM (95% confidence interval range 336–535 nM) and the mean Ki value was 215 nM (95% confidence intervals 168–267 nM).

Time course
A time course was performed using standard reagent concentrations detailed in the protocol with an increased incubation time of 17 h. The assay appeared to be stable for at least 17 h as demonstrated by the curve in Figure 4.

Z’ analysis
Finally, two independent Z’ analysis (4) evaluations were performed, each using 48 replicate values for "total" and NSB wells. DMSO was present at a final concentration of 1% (v/v) in each well. A mean Z’ value of 0.80 was generated. Figure 5 is representative of the data obtained. This is well within the acceptable Z’ value range (0.5–1.0) and confirmed the robustness of the assay.


Conclusion
The adrenergic α2c receptor binding assay has been successfully miniaturized to a 384-well format using the LEADseeker Multimodality Imaging System and is suitable for adaptation to automated screening formats. The assay is tolerant to DMSO up to a concentration of 1% (v/v), robust; achieving a mean Z’ value of 0.80, and has a stability window of at least 17 h.


References
1. Ruffolo, R. R. Jnr et al. Annual Review Pharmacological Toxicology 32, 243–279 (1993).

2. Hein, L. et al. Nature 402, 181–184 (1999).

3. Bylund, D.B. et al. Pharmacological Reviews 46, 121–136 (1994).

4. 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 NK2 neuropeptide receptor binding assay using 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. Screening for potential beta 2-adrenergic receptor antagonists using CypHer5E and IN Cell Analyzer 1000
11. Screening for β2-adrenergic receptor agonists using the pH-sensitive dye,CypHer5, and the IN Cell Analyzer 3000
Post Your Comments:
(Date:12/24/2014)... PharmaBoardroom,s new report, , ... available for free download , digs deep into this ... in the sector today. One area where ... in developing a homegrown pharmaceutical manufacturing base, even if the ... some way off. A cursory comparison with neighboring ...
(Date:12/22/2014)... , Dec. 22, 2014   ... a developer of pathogen-specific therapies for serious ... protecting the microbiome, today announced positive topline ... 1a clinical trial of SYN-004, the Company,s ... of Clostridium difficile (C. difficile) ...
(Date:12/22/2014)... 2014 The American Journal ... original research, reviews and editorials addressing developments and ... today published a provocative article exploring the role ... and potential treatment of prostate cancer. , ... proposes the possibility that there could be a ...
(Date:12/19/2014)... December 19, 2014 BioPlus Specialty ... pharmacies, announces the promotion of Nick Maroulis, Pharm.D. to ... Pharmacy Services. , In this position, Dr. Maroulis ... also managing the directors of our multi-site pharmacies as ... since 1997 and during that time he has served ...
Breaking Biology Technology:PharmaBoardroom Releases New Algeria Pharmaceuticals Report 2PharmaBoardroom Releases New Algeria Pharmaceuticals Report 3Synthetic Biologics Announces Positive Topline Results from Phase 1a Trial of SYN-004 for the Prevention of C. difficile Infection 2Synthetic Biologics Announces Positive Topline Results from Phase 1a Trial of SYN-004 for the Prevention of C. difficile Infection 3Synthetic Biologics Announces Positive Topline Results from Phase 1a Trial of SYN-004 for the Prevention of C. difficile Infection 4Synthetic Biologics Announces Positive Topline Results from Phase 1a Trial of SYN-004 for the Prevention of C. difficile Infection 5Prostate Cancer Experts Dissect the Role of Follicle-stimulating Hormone in the Development, Progression and Potential Treatment of Prostate Cancer 2Prostate Cancer Experts Dissect the Role of Follicle-stimulating Hormone in the Development, Progression and Potential Treatment of Prostate Cancer 3Prostate Cancer Experts Dissect the Role of Follicle-stimulating Hormone in the Development, Progression and Potential Treatment of Prostate Cancer 4BioPlus Specialty Pharmacy Promotes Dr. Nick Maroulis to Vice President of Specialty Pharmacy Services 2
... CHICAGO , May 27 Celsis International, ... its Drug Master Files with the U.S. Food & Drug Administration (FDA) ... solutions and finished goods on the Celsis system. , ... "A growing number of our Rapid Detection customers ...
... Genetics Corporation announced today treatment of the first patient in a nationwide ... have successfully undergone pancreatic surgical resection. , ... "This pivotal study represents one of the largest Phase ... the United States ," said Charles Link , M.D., chairman and ...
... , May 27, 2010 , ... - New Trial Results on ExoSeal(TM) Vascular Closure Device Support its,Clinical ... Cordis ... launch of the,ExoSeal(TM) Vascular Closure Device. ExoSeal(TM) incorporates a number of new,advances in technology ...
Cached Biology Technology:Celsis Rapid Detection's Updated Drug Master Files (DMFs) Accepted by FDA 2NewLink Initiates Pivotal Phase 3 Clinical Trial of HyperAcute(R)-Pancreas Immunotherapy for Resected Pancreatic Cancer 2NewLink Initiates Pivotal Phase 3 Clinical Trial of HyperAcute(R)-Pancreas Immunotherapy for Resected Pancreatic Cancer 3NewLink Initiates Pivotal Phase 3 Clinical Trial of HyperAcute(R)-Pancreas Immunotherapy for Resected Pancreatic Cancer 4CORDIS Launches ExoSeal(TM) Vascular Closure Device 2CORDIS Launches ExoSeal(TM) Vascular Closure Device 3CORDIS Launches ExoSeal(TM) Vascular Closure Device 4
(Date:12/10/2014)... , Dec. 9, 2014 CIE San Diego ... that provides the connective tissue that enhances care coordination ... to easily share client-level information; earned a second $1 ... to organizations serving seniors aging in community and; will ... on December 11 th 4-6p. CIE ...
(Date:12/10/2014)... 9, 2014     ...   Jifflenow, a ... solutions for business-to-business (B2B) events, today announced a ... mobile near-field communication (NFC), Bluetooth low energy (BLE), ... http://photos.prnewswire.com/prnh/20140612/691055) , Jifflenow will integrate its ...
(Date:12/5/2014)... , Dec. 4, 2014  Tute Genomics, a ... $2.3 million in Series A1 funding led by UK-based ... also participated in the investment round. "We ... healthcare community adopts next-generation sequencing and seeks new approaches ... Reid Robison , MD MBA, and CEO of ...
Breaking Biology News(10 mins):Community Information Exchange San Diego Launches Pilot, Secures $1 Million Expansion Grant, and Becomes a "Live Well San Diego" Partner 2Community Information Exchange San Diego Launches Pilot, Secures $1 Million Expansion Grant, and Becomes a "Live Well San Diego" Partner 3Community Information Exchange San Diego Launches Pilot, Secures $1 Million Expansion Grant, and Becomes a "Live Well San Diego" Partner 4Jifflenow And ITN International Bring Cutting-Edge Badge Scanning Technology To B2B Events 2Jifflenow And ITN International Bring Cutting-Edge Badge Scanning Technology To B2B Events 3Tute Genomics Raises $2.3 Million to Scale Up Cloud-Based Genome Analytics and Accelerate Personalized Medicine 2Tute Genomics Raises $2.3 Million to Scale Up Cloud-Based Genome Analytics and Accelerate Personalized Medicine 3
... climate change, a new book by Claire L. Parkinson, ... Center in Greenbelt, Md., can provide some answers. ... Beware the Big Fix," will be published this week ... warming is such a big deal, summarizes 4.6 billion ...
... the Carnegie Institution,s Department of Global Ecology, is among ... the humanities and the arts, business, public affairs, and ... new class of members of the American Academy of ... most prestigious honorary societies, cited Field for his research ...
... pain (or lumbago) is a common ailment often triggered by something ... to remedy the situation? An exercise machine designed specifically for back ... in the Journal of the American College of Sports Medicine ... help the development and endurance of back muscles, you must focus ...
Cached Biology News:NASA scientist publishes new book: 'Coming Climate Crisis? Consider the Past, Beware the Big Fix' 2Carnegie's Chris Field elected to American Academy of Arts & Sciences 2Exercise therapy for low back pain 2