This Userguide will demonstrate measurement of enzyme activity using the Eppendorf BioSpectrometer. In order
to optimize the measurement process, preliminary measurements were performed using the method “single Λ
continuous”. The actual activity measurements were then performed based on these results. Enzyme activities were
determined via linear regression.
For the measurements, a coupled reaction of a hexokinase and a glucose-6-phosphate-dehydrogenase from the
baker’s yeast ‚ was used. Since the Eppendorf BioSpectrometer kinetic is equipped with a temperature controlled
cuvette chamber, temperature dependence of this reaction could also be demonstrated. The highest activity was
detected at 37 °C.
In order to demonstrate the development of a measurement method for a colorimetric assay in the Eppendorf BioSpectrometer, Ponceau-S was scanned across a range of wavelengths from 220 nm to 800 nm to determine its absorbance maxima. Using the novel analysis methods available on the Eppendorf BioSpectrometer, a maximum could be detected at 498 nm using the Eppendorf “SpectraZoom” function.
For the colorimetric assay, a standard curve was generated using Ponceau-S as an example, and measurements were performed. Subsequently, this programmed standard curve was used to calculate an unknown sample concentration.
Fluorescence allows more sensitive and more specific
determination of nucleic acids than the conventional
method of UV-VIS spectroscopy. The present article
describes quantification of dsDNA using the fluorescent
dye PicoGreen®, with special focus on the preparation
of samples for measurement in the Eppendorf
BioSpectrometer, as well as the details of programming.
The methods PicoGreen and PicoGreen-“short” are
compared experimentally. Even though the method
PicoGreen-“short” was measured using only 2
standards, high analytical accuracy was observed.
Photometric measurement of turbidity for the purpose
of determining cell density in a bacterial suspension is
a basic method in every molecular biology laboratory.
In addition to consumption of single use cuvettes and
pipette tips, reduction of the culture volume is an effect
of frequent sample removal and subsequent disposal for
the purpose of measurement.
The present work will show that the alternative use of a
fiber optic probe, which operates without consumables
and without reduction of the bacterial suspension, in
combination with the Eppendorf BioSpectrometer, yields
comparable results and thus presents a good alternative
to a common measurement method.