Galanin receptor binding studies in the membranes of cultured cells measured by fluorescence correlation spectroscopy
✍ Scribed by A. Pramanik; A. Juréus; Ü. Langel; T. Bartfai; R. Rigler
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 65 KB
- Volume
- 13
- Category
- Article
- ISSN
- 0269-3879
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✦ Synopsis
Receptor binding studies with the conventional method require the use of radioactively labelled ligands and include several washing steps. In certain cases, the halflife of the receptor-ligand complex is often shorter or similar to the time required for separation. Interactions between certain ligands (e.g. peptides, natural products) and specific binding sites for their different subtype receptors are, therefore, often not found by conventional receptor binding techniques.
Fluorescence correlation spectroscopy (FCS) is a powerful biophysical tool for examining molecular interactions with very high specificity . In this technique the fluorescence of single dye-labelled molecules excited by a sharply focused laser beam is observed. From the intensity fluctuations due to variations in numbers of molecules in the volume element of observation, the average number of molecules can be directly obtained from the intensity correlation function. Furthermore, from the characteristic correlation times, dynamic processes such as Brownian motion can be analysed . The beauty of FCS is that a mixture of several components with different molecular weights, and corresponding different diffusion times, can be analysed by this technique, without the need for separating unbound components from bound ones . Moreover, measurement can be done in a very tiny volume (5 mL) in a nanomolar range within a very short time (30 s). The small volume element (0.2 fL) allows the detection of single molecules as well as measuring molecular properties at a specific position in a biological structure e.g. in the cell membrane. Recently, we have demonstrated this possibility for the epithelial growth factor interacting with its membrane bound receptors in cultured cancer cells . We have now applied this FCS