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 c ultured cells utilizing Donkey Anti-Rabbit PS SPA Imaging Beads to generate signal in a simple, homogeneous format (Fig 2).

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).

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).

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.

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 for a full list of SPA imaging application references.

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

2. PRICE, E. et al., Direct, quantitative, intracellular measu rement 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


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:10/8/2015)... Celemics, a global pioneer of personalized medicine ... market with the establishment of Celemics America, Inc. ... Europe , has secured itself in the global ... sample preparation for DNA sequencing and genetic testing.  Next ... genetic testing more accurately, quickly, and cost effectively than ...
(Date:10/8/2015)... 8, 2015  The ALS Association, in partnership with ... Challenge to revolutionize communication technology solutions for people living ... ALS (amyotrophic lateral sclerosis) is a progressive neurodegenerative ... and the spinal cord. Eventually, people with ALS lose ... often leads to total paralysis and death within two ...
(Date:10/8/2015)... ... October 08, 2015 , ... ProMIS ... license agreement to develop and commercialize intellectual property rights belonging to The University ... February 2009, and affords exclusive worldwide rights to intellectual property based on use ...
(Date:10/7/2015)... Dallas-Fort Worth, Texas (PRWEB) , ... October 07, ... ... Stem Cell Institute ( ) will present a public educational seminar on ... diseases on Saturday, October 10th from 1:00 pm – 3:00 pm at the ...
Breaking Biology Technology:
... Sciences, Inc. (Nasdaq: CALP ) today announced that ... "Innovation for Experimentation" to be held June 17, 2009 at ... Breakout Session from 2:30 - 4:00 p.m. EDT. , ... by Kevin Hrusovsky, President and CEO, as well as several ...
... BEIJING, June 8 /PRNewswire-Asia/ -- Sinovac Biotech Ltd. (NYSE ... in China, announced today that it,begins the production of a vaccine ... CDC and was delivered to the company on June 8, 2009. ... CEO of Sinovac, commented, ,In,the midst of a global outbreak and ...
... to Help Ease Venipuncture Process , , UnitedPharma ... , COLD SPRING HARBOR, N.Y., June 8 ... Services for America,s Blood Centers (GSABC) bringing the AccuVein ... to GSABC member blood centers. Under the agreement, ...
Cached Biology Technology:
(Date:9/10/2015)... , Sept. 10, 2015 This report ... wearable technology products today, and emerging sensor types that ... risen through the peak of the wearable technology hype ... slide to disillusionment. The common feature with all of ... key enabler for their most useful functions. Sensors collect ...
(Date:9/10/2015)... Sept. 10, 2015 Report Details ... quite delivered upon previous expectations of revenues, consumer adoption ... breakthrough year in which wearables begin to achieve that ... of the main reasons is the entrance of Apple ... the SmartWatches market, but the overall size of the ...
(Date:9/10/2015)... 10, 2015  NXT-ID, Inc. (NASDAQ: NXTD ) ... on the growing mobile commerce market, announces that the ... Touch of Modern, will feature the Wocket® smart wallet ... . Touch Of Modern is ... lifestyle products to its 7.2 million members. Customers are ...
Breaking Biology News(10 mins):
... massive changes to the Earth's landscape , The ice ages ... happening below the ice in the oceans? , Research by ... destruction as whole communities of animals were wiped out by ... it has been thought that these ecosystems somehow dodged extinction ...
... at the Naval Research Laboratory's Marine Meteorology Division ... Monterey Bay Aquarium Research Institute and the National ... of a phenomenon known as the "milky sea." ... ship-based account. This research was published in the ...
... Wright Brothers' first biplanes to monoplanes was an inadvertent ... say two dino flight experts. , According to ... a small early Chinese dinosaur called Microraptor gui used ... to tree in the early Cretaceous. Among the evidence ...
Cached Biology News: