Basics:Experimental Conditions:Specific/Nonspecific

Specific vs. Nonspecific Binding

This section describes the difference between specific and nonspecific binding and between saturable and nonsaturable binding of the radioligand to the tissue. The method used to differentiate between specific and nonspecific are also described. Points 1- 4 are essential in understanding saturation and competition experiments and should be read even if you do not plan to do binding experiments. Points 5 - 7 are designed to demonstrate how to determine the concentration of unlabeled ligand to use to quantitative nonspecific binding. If you are doing or planning on doing receptor binding studies, you are encouraged study points 5 - 7.

1. What is the difference between SPECIFIC and NONSPECIFIC binding?
2. What are some examples of nonspecific binding sites?
3. What is the difference between SATURABLE and NON-SATURABLE binding?
4. When you add radioactive ligand to the tissue preparation it will bind to both specific and nonspecific sites. How can specific and nonspecific binding sites be differentiated in a binding assay?
5. What inhibitor should be used to block binding to specific sites?
6. What concentration of the inhibitor should be used to block the binding of radioactive ligand to specific sites?
The concentration of unlabeled inhibitor to add to block specific binding can be determined by measuring the binding in the presence of increasing concentrations of the unlabeled ligand in the presence of a fixed concentration of radioactive ligand.

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Nonspecific sites may be:

other receptors from the same class. For example [³H]rauwolscine is used to study alpha-2 adrenergic receptors. It binds to alpha-2A, alpha-2B, and alpha-2C subtypes with similar affinities. All three receptor subtypes could be an a specific tissue. If you wanted to study all alpha-2 adrenoceptors without differentiating between subtypes then binding of [³H]rauwolscine to all alpha-2 adrenoceptors would be considered specific binding. If you only wanted to only study alpha-2A subtype, then radioligand bound to the alpha-2A subtype would be considered specific binding and binding to the other subtypes would be considered nonspecific binding.
other receptors from a different class. [³H]rauwolscine binds to serotonin receptors as well as to alpha-2 adrenoceptors. If you want to study alpha-2 adrenoceptors but not serotonin receptors, then binding of [³H]rauwolscine to serotonin receptors would be considered nonspecific binding and binding to the alpha-2 adrenoceptors would be considered specific binding.
binding to tissue protein. The radioligand may bind to tissue protein or become trapped in the lipid membrane. These binding sites would be unrelated to the receptor that you want to study. The binding to the tissue protein would be considered nonspecific.
binding to test tube or glass fiber filters. Many radioligands bind to test tubes and glass fiber filters used to separate bound from free. This binding would be considered nonspecific binding. Binding to test tubes can sometimes be eliminated by choosing the type of test tube (glass or plastic) used to do the binding assay. Binding to glass fiber filters can usually be eliminated to some extent by trying different types of filters from different manufactures or coating the filter with polyethyleneamine which puts a negative charge on the filter.

SATURABLE binding sites are always present in finite amounts. Another way of thinking of this is that if you add enough ligand all of the sites will be occupied with ligand.

NON-SATURABLE binding sites are sites that are present in essentially infinite amounts. No matter how much ligand you add, not all of the sites will be occupied with ligand. The site is non-saturable.

Examples of saturable sites:
- Specific binding sites are always saturable
- Receptors are always saturable binding sites whether they are the site you want to study (specific) or the site you don't want to study (nonspecific)
- Test tubes and glass fiber filters may have saturable, nonspecific binding sites as well as non-saturable binding sites.

Examples of non-saturable sites:
- Low affinity tissue binding sites
- Binding to test tubes and glass fiber filters may be non-saturable

The inhibitor should

be an unlabeled compound
be a competitive inhibitor which is specific for the receptor you wish to study. If you are trying to distinguish between subtypes of a receptor then the inhibitor should have a high affinity for the subtype that you wish to study and a low affinity for any other subtypes.

The inhibitor should not

be the same ligand as the radioactive ligand. If the inhibitor is the same ligand as the radioactive ligand, then it will inhibit the binding of the radioactive ligand at all saturable sites, some of which may be specific and some of which may be nonspecific.
have a high affinity for other receptors (nonspecific, saturable sites) which you do not wish to study.

Most tissue samples will contain multiple sites to which the radioligand can bind. Technology does not currently allow us to directly determine which sites are SPECIFIC Binding Sites.

To determine which sites are specific sites, two sets of tubes are set up. The first set of tubes contain tissue and radioactive ligand. The amount of radioligand bound is referred to as TOTAL BOUND.

Another set of tubes are set up which contain the radioactive ligand, and an unlabeled ligand which will bind to the specific sites, but not to NONSPECIFIC sites. Under this condition the radioactive ligand would be binding to only the nonspecific sites. The amount of radioligand bound under these conditions is referred to as NONSPECIFIC BINDING.

SPECIFIC BINDING is the difference between the Total Binding, and the Nonspecific Binding.

Binding is measured in the presence of the highest concentration of radioactive ligand used in a saturation curve. Under these conditions all of the receptors would be occupied by radioactive ligand.

Binding is also measured in the presence of the same concentration of radioactive ligand plus increasing concentrations of unlabeled ligand.

As the concentration of unlabeled ligand increases the amount of radioactive ligand bound decreases until a plateau is reached. At this point no matter how much unlabeled ligand is added the amount of radioactive ligand bound remains constant.

This residual amount of radioligand bound represents binding to nonspecific, non-saturable sites which can't be displaced by unlabeled ligand.

The amount of inhibitor to use would be the concentration which completely blocks the saturable binding sites. In this example it would be between 10^-9 to 10^-8 M.