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GMS6504 pharmacological Assays Exam With Verified Solutions
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GMS6504 pharmacological Assays Exam With Verified Solutionsbinding assay methods
-radioactivity assay
-fluorescence assay
-polarization assay
-FRET assay
radioactivity assay
-most common way of doing binding assays
-ligand is made radioactive
-levels of radiation in different locations is measured
-pros: easy to measure how much ligand
-cons: ligands bind places we don't want them to bind, washing not 100% efficient
-MUST wash off non-bound ligand
-regulatory hurdles to work with radiation + radiation is EXPENSIVE
-molecular structure stays the same
fluorescence assay
-ligand has fluorescent tag
-pros: does not require radiation, less regulation, cheaper
-cons: ligands bind in places we dont want them to bind, autofluorescence, change in
structure due to fluorescent tag
-wash off non-bound ligand
-adding fluorescent tag to ligand changes its molecular structure = changes in affinity
and efficacy
polarization assay
-measuring polarized fluorescence
-can distinguish fluorescent ligands bound to cell and fluorescent that are not bound
, -polarized light only comes through
-Pros: DO NOT need to wash away unbound ligand, cons: ligand can still bind to different
receptor, small portion of unbound ligand can produce polarized light FRET assay
-looking for fluorescence -take the fluorescent part of molecule and break into 2 pieces =
attach one piece to ligand and other piece to the receptor -when ligand binds to receptor
= 2 parts are held next to each other, which shows fluorescence
-Pros: Doesn't require extra steps- no washing, more specific
-Cons: ?
scintillation proximity
Another type of radioactivity assay
Can be more specific about which radiation we are measuring
scintillation bead
Can measure radiation with scintillation fluid
Put potentially radioactive sample into the fluid= radiation comes off sample and is
absorbed into liquid
Liquid emits visible light= can be measured
Able to discern between non-bound ligand and bound ligand
surface plasmon resonance
-an instrumental technique for measurement of molecular interactions based on changes
of reflectance properties of a sensor coated with an interactive molecule
-gold plate has dextran on it = reactive linker molecule
-attach ligand to dextran
-receptors passed over
-opposite side of gold plate is laser = laser shined
-more angle of reflection changes = more mass = more binding occurs
types of binding experiments
-saturation binding (how much binds)
-displacement binding
-kinetics (how quickly it binds)
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