HM Medical Clinic

Allergyscreen

Application Note PolarScreen Red™ (Invitrogen) Glucocorticoid Receptor Assay
Tecan Infinite™ F500, Fluorescence Polarization The Glucocorticoid Receptor
Assay description
The Glucocorticoid Receptor (GR) belongs to the important Invitrogen has developed a variety of so called PolarScreenTM superfamily of ligand-activated, intracytoplasmatic Nuclear Receptor Assays, for example the Glucocorticoid transcription factors, the so called Nuclear Receptors (NR). Receptor Competitor Assay, in order to offer a simple and NRs mediate cellular responses to a broad range of small reliable method in the research of NRs (3). molecular weight, non-peptide signals, including endogenous hormones and metabolites as well as xenobiotic compounds All these assays include a specific nuclear receptor, a fluorescent Fluoromone ligand and an optimized buffer system; they all work as a competitive system. In the case of How does the Glucocorticoid receptor work? NR-Fluoromone ligand binding a high polarization is the consequence. If the respective compound of interest (any An adequate ligand, for example cortisol, passes the cellular possible ligand) displaces the Fluoromone ligand from the membrane and binds to the cytoplasmatic GR. Due to this complex, the polarization value is lowered to a certain degree. binding the GR releases some heat shock proteins, followed This shift in polarization let one draw conclusions concerning by the release of heat shock chaperones and this yields the the relative affinity of the respective test compound for the NR. free cortisol-receptor subunits. Anyway, these subunits are translocated into the nucleus and work as transcription factors (zinc finger system). The following biological response is cell line specific (1,2). The study of NRs and their ligands is an important field in the development of novel therapeutics to fight especially hormone linked diseases. Application Note Assay protocol 5 μl 3 nM FluoromoneTM GS Red were mixed with 5 μl dexamethasone ready-to-use solution. The reaction was initiated by adding 5 μl 12 nM Glucocorticoid Receptor Working Solution (GR WS). After incubation for 4 h at RT, the fluorescence polarization was measured. For reaction blank, a mix of 5 μl 12 nM GR WS, 5 μl assay buffer and 5 μl dexamethasone dilution buffer was used. The measurements were carried out in five replicates. Figure 1: Scheme of PolarScreenTM NR Assays
As mentioned, the Glucocorticoid Receptor Competitor Assay is an assay to estimate the affinity of some test compounds for the human Glucocorticoid Receptor. This system works with a G factor calculation fluorescent glucocorticoid as ligand (FluoromoneTM GS Red). In the presence of an effective test compound GS Red is G factor calculation was performed using a well containing 5 displaced resulting in decreasing polarization. The system μl of 3 nM GS Red, 5 μl assay buffer and 5 μl dexamethasone works with red fluorescence to minimize background dilution buffer. Reference polarization value: 100 mP, interferences (3). reference blank = same as reaction blank. Measurement settings Material and methods Measurement 1
Instrument
Tecan InfiniteTM F500 filter-based microplate detection system Microplates

Integration time 384 Flat bottom Black Polystyrol small volume micro plates Number of reads (Greiner Bio-One) Manual z-position** calculated from well Reagents and Assay Performance
Table 1: Fluorescence Polarization measurement parameters for
Results and Discussion PolarScreenTM Glucocorticoid Receptor Competitor Assay, Red (Invitrogen P2893). DMSO, dexamethasone diluting buffer (2.5% DMSO in water), Dexamethasone (Sigma, Reagent preparation The assay buffer was prepared according to the manufactors assay protocol and was used for dilution of FluoromoneTMGS Red and Glucocorticoid Receptor stock solutions. Preparation of dexamethasone ready-to-use solutions: 1 mM dexametha- sone DMSO stock solution was initially diluted in DMSO to a range of different concentrated dexamethasone solutions (150 – 0.0015 μM). These solutions were, further on, diluted in H (1/10) and finally, diluted 1/5 with dexamethasone diluting buffer prior to use. Figure 2: Glucocorticoid receptor assay - Titration with the competitor
test compound dexamethasone Application Note The Glucocorticoid Receptor Competitor Assay utilizes the concept of the fluorescence labeled ligand binding to a large receptor. Such receptor-ligand complexes always display high FP values unless the labeled ligand is displaced from the [1] Bran M., Necela and John A. Cidlowski., A Single Amino receptor by a compound, competing for the same receptor Acid Change in the First Zinc Finger of DNA Binding binding site. In that case, the polarization decreases and the Domain of the Glucocorticoid Receptor Regulates shift in polarization value is used to determine the affinity of Differential Promoter Selectivity, J.Biol.Chem. Vol 279, the screening compound for the particular receptor. The 2004, Issue 38, p.39279-39288 greater the affinity of the ligand of interest for the receptor, the lower the polarization value. [2] Stevens A. et al., Dissociation of steroid receptor coactivator 1 and nuclear receptor corepressor recruitment In the present measurements, the reaction was started by to the human glucocorticoid receptor by modification of the addition of GR to a fluorescent glucocorticoid ligand in the ligand-receptor interface: the role of tyrosine 735, Mol. presence of the competitor test compound dexamethasone. Endocrinology, 2003, 17(5):845-859 As expected, increasing concentrations of dexamethasone could subsequently totally prevent the formation of the GS [3] Invitrogen homepage: http://www.invitrogen.com Red/GR complex indicated by the change in the FP signal from 390 to 100 mP (see Figure 1). The inhibition constant, IC50, of 5 nM could be determined and is in accordance with the specification criteria defined by Invitrogen. The IC50 List of abbreviations refers to the dexamethasone concentration which results in a half-maximum shift in polarization value. dimethylsulfoxid The obtained measurement results clearly indicate that the InfiniteTM F500 can easily be optimized to perform fluorescence polarization based receptor-ligand assays, such as described here with the Glucocorticoid Receptor Competitor Assay. Tecan Group Ltd. makes every effort to include accurate and up-to-date information within this publication, however, it is possible that omissions or errors might have occurred. Tecan Group Ltd. cannot, therefore, make any representations or warranties, expressed or implied, as to the accuracy or completeness of the information provided in this publication. Changes in this publication can be made at any time without notice. All mentioned trademarks are protected by law. For technical details and detailed procedures of the specifications provided in this document please contact your Tecan representative. This brochure may contain reference to applications and products which are not available in all markets. Please check with your local sales representative. 2007, Tecan Trading AG, Switzerland, all rights reserved. Tecan® is in major countries a registered trademark of Tecan Group Ltd., Männedorf, Switzerland InfiniteTM F500 is a trademark of Tecan PolarScreenTM is a trademark of Invitrogen Austria T +43 62 46 89 33 Belgium T +32 15 42 13 19 China T +32 15 42 13 19 Denmark +45 70 23 44 50 France +33 4 72 76 04 80
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