Principles of Biological Assays and Lead Compound Identification Flashcards
biochemical assays
identify how compounds interact with an isolated target in an artifical environment
what kind of assay is a receptor binding assay?
biochemical assay
what kind of assay is an enzymatic activity assay?
biochemical assay
what kind of assay looks at protein-protein interactions?
biochemical assay
cell-based assays:
analyze a measurable effect of a given target within the cell, its natural environment
what kind of assay looks at signal transduction pathways?
cell-based assay
an analytical assay looks at
binding –> does your compound bind?
a functional assay looks at
enzyme activity –> does your compound have an effect on the system?
name a protein type that would be difficult to study with a biochemical assay
membrane protein
what quantity of protein is required to do a high throughput screen?
milligram quantities
what are two types of biochemical assays?
analytical and functional
cell-based screens are predominantly _____ assays
functional
to determine binding to a membrane bound receptor, you can
express the receptor of interest in an alternate cell line through DNA construct/ vector and measure binding/ displacement of fluorescent analogue
when would you use a cell-based analytical assay?
when determining binding to a membrane bound receptor
why might you clone a vector into a cell of interest?
to study mutated versions of protein of interest in cell culture, if there is not enough protein of interest in cell of interest, to study cell surface binding for membrane proteins, because reporter output makes for an easier experiment
in a vector, you want the ____ of your protein of interest to cause the transcription of ____
promoter/ reporter protein
for an anti-proliferative assay, you measure _______. If a cell is alive, it converts ____ to ___ via enzymatic reduction. If the cell is dead, no reduction takes place and ____ light is absorbed
the absorption of purple light/ Yellow MTS/ purple formazan/ yellow
Amplex red can be converted to resorufin by horseradish peroxidase in the presence of _______. This is only produced, through a series of reactions, if ____ is made available by the _______ of the protein of interest. If there is no red color, ______
H2O2/ phosphate/ ATPase activity/ the ATPase activity of the protein of interest was inhibited by the drug
chemiluminescence:
substrate absorbs energy from an exothermic chemical reaction. Relaxation to ground state produces quantifiable light emission
bioluminescence:
light is produced through enzymatic activity - luciferase - catalyzes chemical reaction that results in light production
the dual luciferase assay system is a ______ assay
cell-based
in the dual luciferase assay system, the constitutive _______ luciferase serves as a _____
Renilla/ positive control
________ luciferase is only present upon activation of the pathway of interest
firefly
a fluorophore is excited from its ground state by
absorbing a photon