For laboratories that will run a reasonable number of such assays, it is technically simple and inexpensive to synthesize this in the laboratory as outlined below

For laboratories that will run a reasonable number of such assays, it is technically simple and inexpensive to synthesize this in the laboratory as outlined below. use of radioactivity, the resulting method retains a high degree of accuracy and precision, and is suitable for low-cost high-throughput screening. Use of aspects of this method can also improve throughput in other radioimmunoassays. Ginkgolide A Introduction Cyclic AMP (3,5-cyclic adenosine monophosphate; cAMP) is a key second messenger involved in several intracellular signaling pathways (Antoni, 2000; McPhee et al., 2005). Production of cAMP is definitely controlled from the membrane-bound family of adenylate cyclases (ACs) that convert adenosine triphosphate to cAMP. The activity of most of the ACs is definitely regulated by heterotrimeric GTP-binding proteins (e.g., Gs/olf, Gi/o) that directly interact with the intracellular region of GPCRs and may both increase or decrease enzyme activity (Hanoune and Defer, 2001). In addition, phosphodiesterases can catalyze the degradation of cAMP (Weishaar, 1986). The measurement of adenylate cyclase activity can be accomplished Ginkgolide A using radiometric assays that follow the incorporation of a radioactive precursor into cAMP (Salomon, 1979; Schulz and Blum, 1985). More commonly, however, a variety of methods that quantify cAMP have been used both for assessment of adenylate cyclase activity, as well as for measuring tissue content material of cAMP or breakdown of this second messenger. A major advance for the field was the development by Steiner et al. (1972) of a radioimmunoassay (RIA) for cAMP that offered a high degree of level of sensitivity and specificity that was quickly improved by Harper and Brooker (Harper and Brooker, 1975). Efforts at automating this assay actually led to a commercial instrument (Brooker et al., 1976), but this proved unwieldy. More recently, additional methods for quantifying cAMP have used different radiometric or reporter gene strategies (Williams, 2004). Recently developed radiometric assays such as Flashplate technology (NEN/Perkin Elmer) and scintillation proximity assays (SPA, Amersham Biosciences) are based on the competition of [125I]-labeled cAMP and analyte cAMP, resulting in the production of light when the labeled compound is definitely in close proximity to a solid scintillant surface. These assays are easy and reproducible, but are often more expensive than traditional radiometric methods and generally speaking less sensitive. Reporter-gene assays utilize cell lines expressing reporter enzymes such as luciferase, green fluorescent protein (GFP), and -lactamase. Levels of intracellular cAMP are recognized via the manifestation level of a reporter gene that is modulated by transcription element binding to upstream cAMP response elements (CRE). Reporter-gene assay are generally less expensive than the radiometric Rabbit Polyclonal to PKR assays discussed above, however, they are often plagued by high false-positive hit rates. Several novel, non-radiometric methods to quantify cAMP also have recently become available. These assays involve the use of luminescent proximity (ALPHAScreen?) (Ullman et al., 1994), enzyme complementation technology (DiscoveRx, HitHunter? EFC), or electrochemiluminescence (Meso Level Finding) to detect receptor-mediated changes in intracellular cAMP. Each method is definitely readily compatible with automated high throughput screening (HTS), and often demonstrates a high level of level of sensitivity, but requires a high degree of instrumentation to maximize throughput putting it beyond the reach of most academic labs. For this reason, the RIA (or to a lesser degree, protein binding assays using PKA-enriched cells) remains the most widely used technique. Ginkgolide A There has been a recent report detailing an improved procedure for this RIA (Post et al., 2000). Indeed, there are commercial kits available (e.g., Amersham Biosciences) that utilize secondary antibody bound to magnetizable polymer beads, and are separated by magnetic separation or centrifugation. Using the dopamine D1 receptor like a model system, we now describe improvements to this process that decrease the quantity of experimental methods, the assay time, and the assay cost, without sacrificing accuracy or precision. In addition, we describe a rapid method for the routine production of the [125I]-labeled cAMP derivative that is used as the radiomarker with this RIA. Experimental methods and Results Materials and reagents Dihydrexidine was synthesized relating to methods previously published (Brewster et al., 1990). Acetic anhydride, dopamine, IBMX, pargyline, propranolol, SKF38393, and triethyleneamine, and 2-O-monosuccinyladenosine 3:5 monophosphate tyrosyl methyl ester (ScAMP-TME) were purchased from Sigma-Aldrich (St. Louis MO). HEPES was from Study Organics, Inc. (Cleveland OH). Dulbeccos revised eagles press (DMEM), penicillin/streptomycin, and fetal bovine serum (FBS) were purchased from Gibco/Invitrogen. UniFilter-96 GF/B RIA filter plates, Microscint? 20, and Na125I were purchased from Perkin-Elmer (Waltham, MA, USA). Donkey anti-goat antibody was purchased from Jackson ImmunoResearch (Western Grove, PA, USA). Amine terminated BioMag? beads were purchased from Polysciences, Inc. (Warrington, PA, USA), and pre-conjugated Biomagnetic Particles (BMP) to donkey.