What is a receptor binding assays?
Receptor-binding assays have traditionally been a mainstay of drug development. These assays employ a simple biological preparation of cell membranes and a labeled compound to bind specific protein receptors in those membranes.
How do you measure nonspecific binding?
Nonspecific binding is detected by measuring radioligand binding in the presence of a saturating concentration of an unlabeled drug that binds to the receptors. Under those conditions, virtually all the receptors are occupied by the unlabeled drug so the radioligand can only bind to nonspecific sites.
How does radioligand assay work?
They are performed by incubating a range of concentrations of the unlabeled test compound with a fixed concentration of radioligand and measuring the IC50 (nM) of the test compound to competitively inhibit binding of the radiolabeled ligand to its receptor.
What does non specific binding mean?
Nonspecific binding is binding of the assay antibodies which is not correlated with the specificity of the antibodies. Also analytes can bind non-specifically. There are two kinds of nonspecific binding which normally occurs in the lab and which can not be distinguished from each other easily.
What is ligand receptor binding?
Binding of a ligand to a receptor changes its shape or activity, allowing it to transmit a signal or directly produce a change inside of the cell. Stages of signal transduction: ligand-receptor binding, signal relay, response.
What is radioactive binding assay?
Radioactive ligands are commonly used to measure ligand binding to receptors. In this assay, you will measure binding of a radiolabeled ligand to cells or cell membranes containing a receptor of interest. This indicates that virtually every molecule of ligand provided in the stock vial is radiolabeled.
Are receptor specific or nonspecific?
Receptors of a particular type are linked to specific cellular biochemical pathways that correspond to the signal. While numerous receptors are found in most cells, each receptor will only bind with ligands of a particular structure.
Why is non-specific binding linear?
The theory is that under those conditions, virtually all the receptors are occupied by the unlabeled drug, so the radioligand can only bind to nonspecific sites. Nonspecific binding is usually linear with the concentration of radioligand (within the range it is used).
What is radioligand binding used for?
Radioligand binding is widely used to characterize receptors and determine their anatomical distribution, particularly the superfamily of seven transmembrane-spanning G protein-coupled receptors for both established transmitters such as endothelin-1 and an increasing number of orphan receptors recently paired with …
Why does non-specific binding occur?
Among the possible causes of non-specific binding of Abs, the attraction of primary and secondary Abs to endogenous Fc receptors (FcRs) is thought to be the main source of unwanted staining. FcRs are structures on the surface of certain cells that bind the Fc region of Abs.
How do you reduce nonspecific binding in Elisa?
A good blocker should help reduce non-specific binding, but it should also not (or only minimally) react with the antigen, antibodies, or detection reagents. The most common non-ionic detergent blocker is Tween-20. Detergent blockers are cheap, stable, and useful in removing some non-specific binding during wash steps.
When do you need a receptor binding assay?
For these reasons, binding assays are not routinely performed in whole cells. A receptor-binding assay, using only cell components, is generally useful when one wants to determine the affinity of various drugs at a receptor or to determine the number of receptors per cell.
What is the percentage of nonspecific binding in radioligand assay?
In many assay systems, nonspecific binding is only 10-20% of the total radioligand binding. If the nonspecific binding makes up more than half of the total binding, you will find it hard to get quality data.
How is nonspecific binding of a receptor measured?
Nonspecific binding is assessed by wells containing a high concentration (1 μM) of unlabeled SP that successfully competes with the lower levels of the radioactive SP.
How is nonspecific binding of a drug detected?
Nonspecific binding is detected by measuring radioligand binding in the presence of a saturating concentration of an unlabeled drug that binds to the receptors. Under those conditions, virtually all the receptors are occupied by the unlabeled drug so the radioligand can only bind to nonspecific sites.
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