RECEPTOR-LIGAND INTERACTIONS MEASURED BY AN IMPROVED SPUN COLUMN CHROMATOGRAPHY TECHNIQUE - A HIGH-EFFICIENCY AND HIGH-THROUGHPUT SIZE SEPARATION METHOD

Size exclusion chromatography may under the right circumstances be an easy and powerful way to measure in solution the interaction between a receptor and its ligand. Spun column chromatography is a fast size ex clusion technique of increasing popularity, however, little informatio n exists on the method development essential to obtain efficient separ ation in particular when used for analytical purposes. In this paper w e describe a systematic approach to select the optimal parameters for spun column separation including a simple modification of the techniqu e whereby the spun columns are eluted by high-speed gradient centrifug ation. This modification is easy to implement and it considerably impr oves spun column performance. We hypothesize that the high-speed centr ifugation step leads to the release of additional buffer which assists in the complete elution of excluded molecules while the gradient cent rifugation helps to achieve equilibrium across the gel matrix during t he elution. The new method has been used successfully for several diff erent receptor-ligand interactions, and this paper describes a general approach on how to develop new applications of the technique.