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| | Scintillation Proximity Assay (IC50) and PPAR alpha Transactivation Assay (EC50) |
| Description: | Human PPAR alpha receptor was expressed as recombinant glutathione-S-transferase (GST)-fusion proteins in Escherichia coli. The purified GST-hPPAR alpha receptor was used in scintillation proximity assay (SPA)-based receptor-binding assays. Briefly, GST-PPAR receptor was combined in SPA buffer with anti-GST antibodies (AmershamBiosciences, Piscataway, NJ), and a radioligand in assay plates. Yttrium silicate protein A-coated SPA beads (Amersham Biosciences) were added. The assay plates in the presence of varying concentrations of test compounds were incubated with shaking at 15 deg C for approximately 16 h. The plates were then counted in a TopCount scintillation counter (Packard Bioscience, Meriden, CT) to determine the displacement of the radioligand from the receptor by the compound. Nonspecific binding was determined by using a 100-fold excess of the respective unlabeled ligand. The results are expressed as IC50 calculated by a four-parameter logistic equation. EC50 values were determined using transactivation assay. COS-1 cells were cultured, and cotransfected with pcDNA3-PPAR/GAL4 expression vector, pUAS(5X)-tk-luc reporter vector, and pCMV-lacZ as an internal control for transactivation efficiency using Lipofectamine (Invitrogen). Varying concentrations of test compounds were incubated with the transfected cells at 37 deg C for 48 h. Cell lysates were then produced with reporter lysis buffer (Promega, Milwaukee, WI), and luciferase activity in cell extracts was determined by using luciferase assay buffer (Promega) in an ML3000 luminometer (Dynatech Laboratories, Chantilly, VA). |
| Affinity data for this assay | |
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