The stock solution of the enzyme should be prepared freshly for the actual test series and not stored for longer time. To carry out an enzyme assay an aliquot of the assay mixture, e.g. 1 ml, will be transferred into an observation vessel, e.g. a photometric cuvette. The vessel should be connected
with a thermostatting device to achieve rapid warming up. When the assay temperature is reached, the reaction is started by adding the lacking component, e.g. the enzyme. The volume of this last addition should be considered, e.g. if the starter solution comprises 20 µl, only 0.98 ml of the assay mixture is needed to obtain a final assay volume of 1 ml. Mixing is a very crucial task, because the selleck reaction starts immediately after addition, and during a slow mixing and manipulation procedure, e.g. to turn on the instrument, the reaction already proceeds and valuable information may get lost. Therefore mixing must be fast and intense to ensure homogeneous distribution, but any disturbances, like inclusion of air bubbles or dust particles must be avoided. Direct pouring of the solution from the pipette tip into the assay mixture and stirring
with the tip is not advisable, since parts of the solution adhering to the outside surface of the tip will get into the assay and modify the concentration. Disposable stirring sticks are available; the aliquot can be placed on their tip before stirring. Recording of the reaction should start immediately after the last addition and mixing. The reaction should proceed within an appropriate time (between 1 and 5 min), not too fast and not too slow. see more During this time an intense, easily detectable signal should arise. If possible (dependent on the detection
method used) the complete time course (progress curve) of the reaction should be documented; otherwise the reaction is stopped and the signal is measured after a distinct time. For enzyme-catalysed reactions the velocity is directly proportional to the enzyme amount. This rule allows adapting the velocity to the conditions of recording. While for enzyme assays the concentrations of all other components are determined, the amount of enzyme can be varied in PRKD3 order to obtain an optimum reaction course (see next section). The concentration of all substrates and cofactors directly involved in the enzyme reaction should be saturating, so that no component will be rate limiting. The question is, what does “saturating” mean? Binding of these components to the enzyme obeys a hyperbolic saturation function according to the Michaelis–Menten equation (Michaelis and Menten, 1913 and Bisswanger, 2008), i.e. the degree of binding is not directly proportional to the concentration of the component, rather occupation of the binding sites occurs more efficiently at lower concentrations, while with progressive occupation increasing amounts of the component are required.