By Melvin I. Simon
This quantity of equipment in Enzymology covers the present technique for the detection and evaluate of constitutively lively proteins. The chapters written by way of professional authors who're leaders within the field, provide tricks and methods no longer on hand in basic study publications.It is broadly referenced, with worthy figures and tables in the course of the volume.A. specialist authors who're leaders within the box B. generally referenced and priceless figures and tables C. offers tricks and tips to facilitate copy of tools
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Additional info for Constitutive Activity in Receptors and Other Proteins, Part 2
This protein binds to the Gal4 binding sites, interacts with coactivator complexes and increases the expression of the luciferase reporter gene. This enzyme generates light from the D-luciferin substrate. L, ligand. 1. Plasmids construction The first step required cloning of the human SF-1 and the mouse RORA LBDs in-frame with the yeast Gal4 DNA-binding domain (DBD) to yield a chimeric transcription factor able to bind Gal4 binding sites. The yeast Gal4 transcription factor binds specifically through its DBD to a 17-mer DNA sequence that is absent in the genome of higher eukaryotes.
Incubation at room temperature was performed for 15 min. 5. Complexes were added to cells. 6. Plates were incubated at 37 C, 5% CO2, 95% relative humidity. 7. 24 h after transfection, cells were treated with 1 mL of each point of the compound 40Â serial dilution (n ¼ 6 for each dilution point). 125%. 8. Plates were incubated at 37 C, 5% CO2, 95% relative humidity. 9. 24 h later, plates were allowed to equilibrate for 15 min at room temperature and luciferase activity was assayed by adding 25 mL/well BriteLite reagent (Perkin Elmer), incubating 2 min and then reading luminescence using an EnVision Multilabel Plate Reader (Perkin Elmer).
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