Lec5: Measuring receptor binding/signalling Flashcards
What three properties must all radioligands have?
- High affinity, selectivity, specificity
What are the 4 main types of tissues of interest that you would incubate a radioligand with when performing a binding assay?
- Whole cells - culture plate
- Crude homogenenised tissue
- Enriched cell membrane preparations
- Tissue - microscope slides
What is glass fibre filter paper used for?
To separate unbound and bound ligand when performing a binding assay.
What is the main tissue of interest and also the main process of separating bound from free ligands in an assay?
- Crude homogenates
2. Filtration (glass fibre filter paper)
What are the three “reasons” for undertaking Saturation Binding Assays?
- Compare Bmax across different tissues
- Compare binding affinity (Kd) across different species of receptors
- Investigate how receptor mutations affect Kd.
What are the four steps in the procedure for Saturation Binding Assats? CLUE: Step 3 involves dissolving the filter in something.
- Incubate tissue of interest with increasing concentration of radioligand until equilibrium is reached.
- Filtration
- Dissolve filter in scintillation fluid
- Count bound radioactivity
Why is non-specific binding non-saturable, and therefore how does it increase as more radioligand concentration is increased?
Because it binds to cell membrane, which is a huge space near impossible to completely cover in radioligands, so as radioligand conc. is increased, non-specific binding also increases linearly.
What are the two “types” of non-specific binding?
- Ligand distributes into llipid components, which “stick” to any surface.
- Free ligand that escaped separation/filtration phase.
How is specific binding distinguised from non-specific binding? Include diagrams and graphs.
- Work out total binding using radioligand only
- Work out non-specific binding by incubating radioligand with excessive conc. of non-radioactive displacer ligand and tissue of interest, and measure bound radioactivity (gives NSB)
- Specific binding = Total binding - NSB
NOTE: Can work out both Kd and Bmax from the graph
Why is non-specific binding a LINEAR line when graphed?
Because it is non-saturable.
Why would a Competition Binding Assay be used in place of a Saturation Binding Assay?
SBA only possible when radiolabelled form of drug is available, but radiolabelling is very expensive. So instead screen the unmarked drug against a known, highly specific radioligand.
How does the Law of Mass Action apply to binding assays? (And what do shallow and steep curves indicate respectively?)
An 81 fold increase in the concentration of unlabelled drug should cause the concentration of bound radioligand to fall from 90% to 10% (ie. in the space of two log units).
Steep = drug undergoes allosteric interaction with receptor
Shallow = drug binds to other receptors
What is IC50? Relate the value of IC50 to the strength of the inhibitor.
- Concentration of inhibitor at which binding of ligand is reduced by half.
- Smaller IC50 = Stronger inhibitor
Why does IC50 compare relative affinity, not absolute affinity? That is, why does IC50 not equal Kd?
Because the value of IC50 changes depending on the amount of radioligand that was initially used (due to competition). That is, if 10x more radioligand is used, 10x more unlabelled drug is required to displace/outcompete it, and therefore IC50 increases.
What is the disassociation constant? Write the equation for it, and explain how this is relevant to if the experiment is carried out at a concentration of radioligand below or near its Kd.
The concentration of unlabelled drug which occupies 50% of receptor population in the absence of the radioligand.
