Navigation Links
Utilization of Donkey Anti-Rabbit PS SPA Imaging Beads in the quantitative measurement of cAMP in cells

Key words: SPA Imaging Beads • LEADSeeker • cAMP • receptor binding assay


SPA Imaging Assays are homogeneous immunoassays based on the coupling of one assay component to an SPA Imaging Bead in order to generate a signal. Polystyrene (PS) SPA Imaging Beads coated with donkey anti-rabbit antibody enable the immobilization of rabbit secondary antibodies during the assay. The Donkey Anti-Rabbit PS SPA Imaging Beads bind to the Fc region of the secondary antibody, leaving the antigen binding Fab region unhindered. Application data is demonstrated here for the utilization of Donkey Anti-Rabbit PS SPA Imaging Beads in a cAMP assay.


SPA Immunoassay principle
Binding radiolabeled ligands to antibodies immobilized on the surface of an SPA Imaging Bead brings the radiolabel into close proximity with scintillant incorporated within the bead. The emitted radiation (beta particles for tritium or Auger electrons for iodine-125) then stimulates the scintillant to emit light (Fig 1). Unbound radiolabeled ligand is not in close enough proximity to the scintillant for energy transfer to occur and no signal is generated. Light emitted by stimulated SPA Imaging Beads can be detected by platforms such as the LEADseeker™ Multimodality Imaging System.


cAMP assay
Adrenergic receptors are linked to adenylate cyclase via either inhibitory or stimulatory G proteins. These receptors, when activated by agonist or antagonist occupancy of ligand binding sites, affect the intracellular generation of cAMP. In response to receptor binding, adenylate cyclase converts AMP to cAMP, which exerts its effect by activating a protein kinase capable of phosphorylating specific substrates.

Here, we describe a direct screening 384-well assay that enables the direct extraction and assay of cAMP from cultured cells utilizing Donkey Anti-Rabbit PS SPA Imaging Beads to generate signal in a simple, homogeneous format (Fig 2).


Materials
Products used

LEADseeker Multimodality Imaging System 18-1140-71

Donkey Anti-Rabbit PS SPA Imaging Beads RPNQ0299


Other material required
Assay buffer

cAMP standard

Adenosine 3',5'-cyclic phosphoric acid 2'-0-succinyl-3-[125I] iodotyrosine methyl ester, 147.5 kBq, 4 µCi, lyophilized.

Rabbit anti-succinyl cAMP serum, lyophilized.

Lysis reagent 1

Lysis reagent 2

The above reagents are available in the cAMP – [125I] Direct Biotrak™ SPA Screening Assay (RPA 559).


Method
Chinese hamster ovary (CHO) or A431 cells were seeded into 384-well cluster plates suitable for both cell culture and imaging. Cells were stimulated (20 min 37 °C, 5% CO2, 95% humidity) either with forskolin (1–1000 µM) (Fig 3), one of the β-adrenoreceptor agonists: isoproterenol, epinephrine, salbutamol, or noradrenaline (1.25–1000 nM) (Fig 4), or pre-incubated for 30 min with the non-selective adrenoreceptor antagonist propranolol (0.001–1000 nM), before stimulation with isoproterenol (1000 nM) (Fig 5) (1).


A two-stage method was applied, where the cells were cultured and lyzed in a separate vessel. The culture supernatant was gently aspirated and the cells lyzed with 0.25% (w/v) dodecyltrimethyl ammonium bromide. An aliquot of lysate was transferred to a second plate for assay.

Working standards (0.2–25.6 pmol/well) were pipetted into empty wells of the plate. Equal volumes of antisera (20 µl/well), [125I]cAMP (~ 35 000 cpm/well), and Donkey Anti-Rabbit PS SPA Imaging Beads (20 µl, 0.4 mg/well) were added to the microplate wells containing standards and samples. The plates were sealed and incubated at room temperature for 15–20 h, without agitation. The amount of [125I]cAMP bound to the SPA Imaging Beads was determined by imaging on the LEADseeker Multimodality Imaging System for 5 min (2).


Results
The results in Figure 3 show the cAMP produced by CHO cells in response to stimulation by forskolin, an activator of adenylate cyclase. The cells have responded in a dose dependent manner and an increase in cAMP levels can be detected at 1 pmol forskolin, the lowest concentration used.

Figures 4 and 5 show the measurement of cAMP in A431 cells treated with known B2 adrenergic agonist and antagonists. The EC50 values obtained are comparable to those quoted in the literature (3), showing that this imaging assay in 384-well format can be successfully used for high-throughput assays.


Conclusion
Donkey Anti-Rabbit PS SPA Imaging Beads give a good specific signal and large signal window in the cAMP assay. These beads have been utilized in pharmacological experiments and the results correlate very well with other coated SPA beads and non-SPA formats.

Please refer to the SPA imaging bibliography at www.amershambiosciences.com/LEADseeker for a full list of SPA imaging application references.


References
1. CHIESI, M. et al., Trends in Pharmacol. Sci., 22, (5), 247–254, (2001).

2. PRICE, E. et al., Direct, quantitative, intracellular measurement of cyclic AMP in cells: A LEADseeker assay utilizing Protein A Polystyrene SPA Imaging Beads. Poster presentation, Screening Europe Conference, London (2004).

3. SCHWARTZ, M.W. et al., Nature, 404, 661–671, (2000).



back to top
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Optimized Imaging of Protein Gels Stained with Coomassie Brilliant Blue Dye
2. Detect Released Oligosaccharides Using the Eagle Eye II Still Video Imaging System
3. Imaging Chemiluminescent Western Blots
4. Eagle Eye Optical Filter Optimized for Imaging Silver-Stained Gels
5. Imaging and Analysis of DIGE Product Information Sheet, Rev B
6. The development of a CXCR2 chemokine receptorbinding assay using the LEADseeker Multimodality Imaging System
7. Performance of the AMARA HitHunter Chemiluminescence Kinase Assay with the LEADseeker Multimodality Imaging System
8. The development of a melanocortin MC4 receptor binding assay using the LEADseeker Multimodality Imaging System
9. The development of a melanocortin MC5 receptor binding assay using the LEADseeker Multimodality Imaging System
10. The development of a calcitonin-gene related peptide (CGRP) receptor-binding assay using the LEADseeker Multimodality Imaging System
11. Development of a histamine H1 receptor binding assay using the LEADseeker Multimodality Imaging System
Post Your Comments:
(Date:7/11/2014)... 11, 2014 , Not for release, ... or from any jurisdiction where to do so would constitute ... Shire plc ("Shire" or the "Company") (LSE: SHP, NASDAQ: ...  Shire confirms it has held a meeting with representatives of ... without the prior agreement or approval of AbbVie. ...
(Date:7/11/2014)... 2014 /CNW/ - Sunovion Pharmaceuticals Canada Inc. (Sunovion) today ... acetate) for use as a once-daily adjunctive therapy for ... who are not satisfactorily controlled with conventional therapy.  APTIOM ... under 18 years of age. Epilepsy ... according to Epilepsy Canada, it affects 0.6% of the ...
(Date:7/10/2014)... 2014 Product and process impurities ... abundance and are often “lost in the noise,” ... Join presenters Dr. Rowel Tobias, Senior Scientist, Protein ... Head of Quality at Nanotherapeutics, Inc., to lear ... speed detection and quantitation while achieving the required ...
(Date:7/10/2014)... CA (PRWEB) July 10, 2014 Unraveling ... closer together. The Archer Family purchased a ... within DNA, but ended up with a lifetime of ... contained within the family members’ DNA genuinely brought the ... decided to search deeper into genetic history and prior ...
Breaking Biology Technology:Shire plc - Statement re: Media Speculation 2Sunovion Pharmaceuticals Canada Inc. Announces Health Canada Approval of APTIOM(TM) (eslicarbazepine acetate) as a Once-Daily Adjunctive Treatment for Partial-Onset Seizures in Patients with Epilepsy 2Sunovion Pharmaceuticals Canada Inc. Announces Health Canada Approval of APTIOM(TM) (eslicarbazepine acetate) as a Once-Daily Adjunctive Treatment for Partial-Onset Seizures in Patients with Epilepsy 3Sunovion Pharmaceuticals Canada Inc. Announces Health Canada Approval of APTIOM(TM) (eslicarbazepine acetate) as a Once-Daily Adjunctive Treatment for Partial-Onset Seizures in Patients with Epilepsy 4Novel Ms Techniques Speed Detection of Biopharmaceutical Product and Process Impurities, New Webinar Hosted by Xtalks 2Ancestry DNA Test Reveals a Controversial and Undiscovered Family History 2Ancestry DNA Test Reveals a Controversial and Undiscovered Family History 3Ancestry DNA Test Reveals a Controversial and Undiscovered Family History 4Ancestry DNA Test Reveals a Controversial and Undiscovered Family History 5
... Fusion Platform Includes Innovative Biometric ... ... 12 WCC Smart Search & Match,a leading provider of ... Systems, a leader in providing,innovative eye biometric technology for identification ...
... Throughout a ... Product,s Lifecycle, NEW YORK, Feb. ... an industry leader in patient,enrollment for clinical trials, will join the ... this partnership, Ketchum,will greatly expand the depth of services offered to ...
... (NYSE: BMY ) today announced that the company,s ... the Board. Mr. Cornelius, who,was named chief executive officer ... well. Mr. Cornelius, current employment contract, which,expires in May ... B.,Campbell, chairman, president and CEO, Textron Inc., as lead ...
Cached Biology Technology:WCC Smart Search & Match and Retica Announce Global Collaboration 2Ketchum Joins Forces with Clinical Trial Recruitment Agency MMG 2Ketchum Joins Forces with Clinical Trial Recruitment Agency MMG 3Ketchum Joins Forces with Clinical Trial Recruitment Agency MMG 4Bristol-Myers Squibb Board Elects James M. Cornelius Chairman of the Board 2
(Date:7/11/2014)... Tenn., July 11, 2014--Researchers at the Department of ... R&D 100 awards, presented by R&D Magazine in ... , "These awards recognize the tremendous value of ... Moniz. "Research and development at the National Labs ... challenges and pursue the scientific and technological innovations ...
(Date:7/11/2014)... . X-ray ... of X-rays through a specimen instead of attenuation resulting ... often of much higher quality than those based on ... Pfeiffer are particularly interested in developing new approaches for ... imaging. One main goal is to make this method ...
(Date:7/11/2014)... Social animals often develop relationships with other group members ... In wild chacma baboons the strategy for grooming activities ... are just published in the scientific journal Biology ... of baboons is not practiced without ulterior motives. To ... for the individual, while grooming another individual can provide ...
Breaking Biology News(10 mins):ORNL wins eight R&D 100s 2ORNL wins eight R&D 100s 3ORNL wins eight R&D 100s 4New simple setup for X-ray phase contrast 2Baboons groom early in the day to get benefits later 2
... in German . There are many ... But how can the chemical properties of a material be ... of Technology developed a method to attach molecules at exactly ... can allow the positioning of chemical signals, telling living cells ...
... benefits of breast milk have long been appreciated, but ... a unique property that makes mother,s milk better than ... The finding, published in the August issue of ... explains how breast milk, but not infant formula, fosters ...
... it out to regulate gene expression on the DNA ... enzymes that transcribe DNA into RNA. Their repressor opponents ... coil around histone proteins, an amalgam called chromatin, effectively ... RNA strand. Both teams maneuver by chemically modifying ...
Cached Biology News:The laser beam as a '3-D painter' 2Breast milk promotes a different gut flora growth than infant formulas 2Breast milk promotes a different gut flora growth than infant formulas 3Controlling gene expression: How chromatin remodelers block a histone pass 2Controlling gene expression: How chromatin remodelers block a histone pass 3
... J. Davison and Richard ... Contents include: information on ... paramyxoviruses, influenza and immunodeficiency ... analysis of viral NA ...
...
...
...
Biology Products: