Bioluminescence: Fundamentals and Applications in by Gérald Thouand, Robert Marks

By Gérald Thouand, Robert Marks

This ebook evaluate sequence provides present traits in smooth biotechnology. the purpose is to hide all features of this interdisciplinary know-how the place wisdom, equipment and services are required from chemistry, biochemistry, microbiology, genetics, chemical engineering and laptop technological know-how. Volumes are geared up topically and supply a accomplished dialogue of advancements within the respective box over the last 3-5 years. The sequence additionally discusses new discoveries and functions. certain volumes are devoted to chosen issues which concentrate on new biotechnological items and new approaches for his or her synthesis and purification. quite often, specified volumes are edited through famous visitor editors. The sequence editor and writer will even if continually be happy to obtain feedback and supplementary info. Manuscripts are authorized in English.

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Extra resources for Bioluminescence: Fundamentals and Applications in Biotechnology - Volume 2

Example text

Resuscitation will normally allow them to build up the ATP pool again or they will die. 3 Extraction of Intracellular ATP The extraction of intracellular ATP has two functions: (1) to open up the cell walls or membranes to release ATP; and (2) to inactivate all enzymes participating in forming or degrading ATP [1]. Because the turnover is so rapid (see Sect. 4), the inactivation should be immediate. The extractant must penetrate the cell walls and membranes to inactivate the enzymes. This means that ATP is simultaneously released.

2 ATP Assays In ATP assays, the choice of luciferase is affected by the following parameters: 1. The number of ATP molecules consumed per emitted photon affects the sensitivity and decay rate. Quantum efficiency should therefore be high. 2. The turnover number should be as high as possible. 3. The Km value for ATP affects the linear range. From the Michaelis–Menten equation (Eq. 2), v/V = S/(S + Km), we see that S must be negligible compared to Km to give absolute linearity. A low Km is therefore a disadvantage when measuring high concentrations of ATP.

Luciferase may be inactivated by the walls of the cuvette or microplate well, proteases in the sample, or detergents used for extracting bacteria. Product inhibition should not appear when the reagent is optimized for analytical purposes. Under conditions when luciferase is not inactivated or subjected to an increasing inhibition as a consequence of accumulating product inhibition, the intensity of the light emission at low substrate concentrations is proportional to the substrate concentration.

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