IPSIndian Journal of Pharmacology
Home  IPS  Feedback Subscribe Top cited articles Login 
Users Online : 598 
Small font sizeDefault font sizeIncrease font size
Navigate Here
  Search
 
 » Next article
 » Previous article 
 » Table of Contents
  
Resource Links
 »  Similar in PUBMED
 »  Search Pubmed for
 »  Search in Google Scholar for
 »Related articles
 »  Article in PDF (131 KB)
 »  Citation Manager
 »  Access Statistics
 »  Reader Comments
 »  Email Alert *
 »  Add to My List *
* Registration required (free)

 
In This Article
 »  Abstract
 »  Acknowledgments
 »  References
 »  Article Figures

 Article Access Statistics
    Viewed3816    
    Printed113    
    Emailed4    
    PDF Downloaded182    
    Comments [Add]    
    Cited by others 2    

Recommend this journal

 


 
CASE REPORT
Year : 2008  |  Volume : 40  |  Issue : 2  |  Page : 89-90
 

Demonstration of LanthaScreen™ TR-FRET-based nuclear receptor coactivator recruitment assay using PHERAstar, a multi-detection HTS microplate reader


1 Bioassay Research Laboratory, Natural Remedies Pvt Ltd, 5B Veerasandra Industrial area, Hosur Road, Bangalore - 560 100, India
2 BMG LabTech Pty Ltd, P.O. Box 469, Mount Eliza, Victoria 3930, Australia

Correspondence Address:
Prashanth C.G D'Souza
Bioassay Research Laboratory, Natural Remedies Pvt Ltd, 5B Veerasandra Industrial area, Hosur Road, Bangalore - 560 100
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0253-7613.41046

Rights and Permissions

 » Abstract 

An attempt was made to demonstrate the possibility of performing LanthaScreen™ TR-FRET based nuclear receptor coactivator recruitment assay using PHERAstar, a multi-detection HTS microplate reader. LanthaScreen™ nuclear receptor coactivator recruitment assay (M/s Invitrogen corporation, USA) was performed using PPAR-gamma receptor preparation in the agonist mode. TR-FRET measurements were done on PHERAstar, a multimode microplate reader (BMG LABTECH, Germany). The Lanthascreen PPAR gamma coactivator recruitment assay was successfully performed in the PHERAstar, multimode microplate reader. This was evidenced by an assay robustness score (Z') of 0.71. The current work demonstrates the suitability of using PHERAstar, a multi-detection HTS microplate reader.for performing LanthaScreen™ TR-FRET based nuclear receptor coactivator recruitment assays.


Keywords: HTS, LanthaScreen TM nuclear receptor coregulator assays, nuclear receptors, PHERAstar, time resolved-fluorescence resonance energy transfer


How to cite this article:
D'Souza PC, Ally N, Agarwal A, Rani P, Das M, Sarma D. Demonstration of LanthaScreen™ TR-FRET-based nuclear receptor coactivator recruitment assay using PHERAstar, a multi-detection HTS microplate reader. Indian J Pharmacol 2008;40:89-90

How to cite this URL:
D'Souza PC, Ally N, Agarwal A, Rani P, Das M, Sarma D. Demonstration of LanthaScreen™ TR-FRET-based nuclear receptor coactivator recruitment assay using PHERAstar, a multi-detection HTS microplate reader. Indian J Pharmacol [serial online] 2008 [cited 2020 Aug 12];40:89-90. Available from: http://www.ijp-online.com/text.asp?2008/40/2/89/41046


Nuclear receptors (NRs) are a superfamily of ligand-activated transcription factors that mediate a wide range of cellular responses. The endogenous ligands for NRs include steroidal hormones, eicosanoids, retinoids, thyronines, bile acids, and other metabolites. [1] NRs influence important biological effects, such as growth, metabolism, and reproduction, and are recognized as important therapeutic targets. [2] High-throughput screening methods for nuclear receptor ligands include nuclear receptor binding assays, nuclear receptor coregulator assays, and nuclear receptor functional cell-based assays. Nuclear receptor coregulator interaction assays are an important class of cell-free functional assays. These assays are based on the principle that nuclear receptors recruit/displace coregulatory peptides upon ligand activation. One of the important assay technologies available for the study of NR coregulator recruitment is called time resolved-fluorescence resonance energy transfer [3] (TR-FRET).

In TR-FRET assays, the biomolecular interaction is detected by energy transfer between two fluorophores that are deliberately employed in these assays. The fluorescence energy transfer results in dual emissions, which can be expressed as a ratio and used as the assay signal. This ratiometric measurement is helpful to counteract assay interference like quenching exhibited by test samples. Besides the ratiometric nature of these assays, TR-FRET also utilizes the phenomenon of time-resolved fluorescence (TRF). TRF is a phenomenon exhibited by chelates of the lanthanide group of elements (europium, samarium, terbium, etc.) Upon excitation, the fluorescence that is emitted by the lanthanide chelate does not decay spontaneously like commonly employed fluorophores, instead there is delayed decay of fluorescence. Autofluorescence of test compounds/microwell plates/buffers (background fluorescence) is the bane of bioassays. The decay time of the background fluorescence falls within the decay time of commonly employed fluorophores in assay systems and thus there can be assay interference. By utilizing lanthanide chelates, the signal measurement window can be set at a much longer time interval which will be free from background autofluorescence. Thus, the TR-FRET method combines the benefits of ratiometric measurement and the TRF phenomenon. [4]

Invitrogen, USA, offers a TR-FRET-based nuclear receptor coactivator assay platform under the trade name LanthaScreen TM . In the current study an attempt is made to demonstrate the possibility of performing the LanthaScreen TM nuclear receptor coregulator assay using the PHERAstar, a multilabel plate reader from BMG LABTECH, Germany. LanthaScreen TM nuclear receptor assays using the PHERAstar have not so far been reported in the literature. This demonstration would validate the compatibility of PHERAstar instrumentation for LanthaScreen TM nuclear receptor coregulator assays. This is required since advanced assay methodologies like TR-FRET require appropriate instrumentation for proper signal measurement. In the current study, the LanthaScreen TM peroxisome proliferator activating receptor-gamma (PPAR-gamma) coactivator recruitment assay (agonist mode) is employed. The assay principle is described in [Figure 1].

In brief, the assay procedure was as per Invitrogen's kit insert (catalog # PV 4548). All the reagents were procured from Invitrogen, except rosiglitazone, which was obtained from Cayman Chemicals, USA. The assay was performed in 384 black microwell plates (Corning # 3676). A 20 µl total assay reaction included 5 nM GST-tagged PPAR-gamma receptor (glutathione S transferase-tagged PPAR-gamma receptor) 125 nM of coregulator peptide, 5 nM of TB-anti-GST-tagged antibody (terbium-anti-glutathione S transferase tagged), 5 mM DTT (dithiothreitol), and varying concentrations of rosiglitazone (a known agonist for PPAR gamma receptor) in the assay buffer supplied by Invitrogen. The negative control was devoid of the agonist but contained everything else contained in the agonist wells. Following 1-h incubation in the dark, TR-FRET measurements were made in the PHERAstar using the following settings: optical module - LanthaScreen TM , delay time - 100 µsec, and integration time - 200 µsec. The ratiometric emission (520/490) was plotted against varying ligand concentrations. The data was analyzed using GraphPad Prism, version 4.00 for Windows (GraphPad Software, San Diego, California, USA) using the sigmoidal curve equation with variable slope to obtain EC 50 values [Figure 2]. The assay quality/robustness score - Z' was calculated as per Zhang [5] et al . and was found to be 0.71 (a value above 0.5 indicates a very robust assay). From the observed data, the PHERAstar, a multilabel plate reader from BMG Labtech, Germany, is suitable for LanthaScreen TM nuclear receptor coregulator assays.


 » Acknowledgments Top


  1. Cynthia Wadsworth, PhD, Senior Technical Support Specialist for Drug Discovery Products from Invitrogen Corporation (Carlsbad CA, USA) is thanked for her support in helping to make this work possible
  2. Department of Science and Technology, Government of India, for financial assistance.


 
 » References Top

1.Bain DL, Heneghan AF, Connaghan-Jones KD, Miura MT. Nuclear receptor structure: Implications for function. Ann Rev Physiol 2007;69:201-20.  Back to cited text no. 1    
2.Hansen MK, Connolly TM. Nuclear receptors as drug targets in obesity, dyslipidemia and atherosclerosis. Curr Opin Investig Drugs 2008;3:247-55.  Back to cited text no. 2    
3.Pearce KH, Iannone MA, Simmons CA, Gray JG. Discovery of novel nuclear receptor modulating ligands: An integral role for peptide interaction profiling. Drug Discov Today 2004;9:741-51.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.Seethala R. Homogenous assays for high-throughput and ultrahigh-throughput screening. In: Seethala R, Fernandes PB, editors. Handbook of drug screening. New York: Dekker; 2001. p. 69-128.  Back to cited text no. 4    
5.Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4:67-73.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]


    Figures

  [Figure 1], [Figure 2]

This article has been cited by
1 Mechanism-based testing strategy using in vitro approaches for identification of thyroid hormone disrupting chemicals
AlberTinka J. Murk,Eddy Rijntjes,Bas J. Blaauboer,Rebecca Clewell,Kevin M. Crofton,Milou M.L. Dingemans,J. David Furlow,Robert Kavlock,Josef Köhrle,Robert Opitz,Theo Traas,Theo J. Visser,Menghang Xia,Arno C. Gutleb
Toxicology in Vitro. 2013; 27(4): 1320
[Pubmed] | [DOI]
2 A Selective Luminescent Probe for the Direct Time-Gated Detection of Adenosine Triphosphate
Evan A. Weitz,Jennifer Y. Chang,Adam H. Rosenfield,Valerie C. Pierre
Journal of the American Chemical Society. 2012; 134(39): 16099
[Pubmed] | [DOI]



 

Top
Print this article  Email this article

    

Site Map | Home | Contact Us | Feedback | Copyright and Disclaimer
Online since 20th July '04
Published by Wolters Kluwer - Medknow