Basics:Experimental Conditions

Experimental Conditions Used in Receptor Binding Studies

Factors to consider in designing receptor binding experiments.

This page explains basic experimental conditions that need to be considered in doing receptor binding studies. You are encouraged to read points 1 through 4 as they are essential in understanding how saturation experiments and competition experiments are conducted. You are encouraged to read points 5 if you are actually doing or planning on designing new receptor binding studies.

1. Identify an appropriate radioactive ligand to use for the experiment.
2. Tissue preparation to be used in the experiment.
3. Identify a method for separating bound from free.
4. Identify a method for distinguishing specific from nonspecific binding.
5. Appropriate assay conditions:
a. pH of the assay buffer
b. Buffers used
c. Additional reagents that may be required for binding
d. Temperature of the binding reaction

Continue to the beginning of the Saturation Experiments

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The tissue could be:

cell membranes isolated from cells grown in culture.
cell membranes from organ tissues that have been washed several times to remove any endogenous ligand.
intact cells in tissue culture.
organ slices (example brain) where binding is studies using autoradiography.
soluble receptor that is present in tissue or membrane receptor that has been solubilized.

The affinity of a ligand for a receptor generally varies with the pH. Generally using a physiological pH, such as 7.4, is best so that the results are comparable to what is seen in vivo.

The most common buffers used in saturation experiments are Tris, Hepes, sodium phosphate and glycylglycine. Buffers can affect the binding of radioligand to the receptor (Deupree, 1997). If possible, using the same buffer as for assays measuring response is best so that the results can be compared. Using the same buffer as used by other laboratories studying the same receptor may be helpful so that the results will be comparable.

Additional reagents are sometimes required for binding of radioactive ligands to some receptors. MgCl₂ is used in many receptors binding assays. Initially this was used to help precipitate the radioligand receptor complex during centrifugation. Many investigators include MgCl₂ so that their experimental conditions will be similar to that reported by others. MgCl₂ may help to convert the receptor to a form that has a higher affinity for agonists.

NaCl is often used in adrenergic receptor binding studies to convert the receptor to a form that has a lower affinity for agonists.

GTP or its non-hydrolyzeable analog (Gpp(NH)p) are often used when agonist binding is also going to be studied in subsequent competition experiments. Gpp(NH)p converts the receptor into a form that has a low affinity for agonist.

Most binding assays can be conducted at room temperature. However, proteolytic enzymes may degrade peptide ligands if the assay is run at room temperature. Some assays work better at 4^oC than at room temperature, and sometimes the affinity of the radioligand for the receptor is higher at 4^oC than at 25^oC . It takes longer to reach steady-state when the reaction is run at 4^oC than at room temperature.