A micro-volume adaptation of a stopped-flow system; use with μg quantities of muscle proteins

Abstract

Stopped-flow spectroscopy is a powerful method for measuring very fast biological and chemical reactions. The technique however is often limited by the volumes of reactants needed to load the system. Here we present a simple adaptation of commercial stopped-flow system that reduces the volume needed by a factor of 4 to ≈120 μl. After evaluation the volume requirements of the system we show that many standard myosin based assays can be performed using <100 μg of myosin. This adaptation both reduces the volume and therefore mass of protein required and also produces data of similar quality to that produced using the standard set up. The 100 μg of myosin required for these assays is less than that which can be isolated from 100 mg of muscle tissue. With this reduced quantity of myosin, assays using biopsy samples become possible. This will allow assays to be used to assist diagnoses, to examine the effects of post translational modifications on muscle proteins and to test potential therapeutic drugs using patient derived samples.

Keywords: Actin; Ca regulation; Muscle biopsies; Myofibrils; Myosin; Rapid mixing; Troponin.