shorter wavelengths is attenuated and does not enter the
detector (photomultiplier tube). This is beneficial for FRET
experiments whereby one excites at a lower wavelength and
wants to record the emitted light from the acceptor fluorophore. Therefore, having a long-pass filter separating the donor
excitation and acceptor emission wavelengths will prevent any
scattered light of the incident wavelength or FRET donor
emission from contributing to the fluorescence signal detected
by the instrument. Other filters can be used such as band-pass
filters to detect only a specific range of light depending on
experimental conditions. For a good example of this see Gzyl
and Wieden [9].
13. The xenon arc lamp needs to reach operating temperature prior
to the first measurement to achieve a stable signal (typically
15 min prior to the first measurement).
14. Each syringe holds one reaction solution, generally keeping the
two molecules of interest separate before mixing. Upon mixing, the solutions dilute each other by half if both syringes are
the identical volume, thus the final concentration of each reactant in the cuvette will be half that of the individual reaction
solutions before mixing. Therefore, it is important to prepare
reaction solutions that are double the desired reaction concentration. Some stopped-flow instruments have three or more
syringes so that three different reactants can be rapidly or
sequentially mixed without having to incubate two with each
other prior, and as such, one must be careful with the dilution
upon mixing three syringes and the desired concentrations of
each reactant in the cuvette. Additionally, the syringes can be of
different sizes having different diameters such that upon
moving the drive piston an unequal volume of solution in
either syringe is mixed resulting in a greater dilution than 1:1
which can be beneficial depending on the experiment. Protein
refolding experiments tend to utilize stopped-flow experimental setups with different syringe sizes such as in Visconti
et al. [10].
15. The incubation temperature is dependent on the particular
experiment and/or molecular system in the experiment and
thus can be adjusted to suit the respective experimental
conditions.
16. It is important to centrifuge each reaction solution at maximal
speed in a tabletop microcentrifuge before loading into the
stopped-flow to remove any precipitate. Precipitates can clog
the lines of the stopped-flow and/or interfere with subsequent
experiments if not cleaned properly. Furthermore, any precipitate that is not removed and part of a mixed sample will scatter
light during detection, thus interfering with the measurement.
284
Harland E. Brandon and Hans-Joachim Wieden
detector (photomultiplier tube). This is beneficial for FRET
experiments whereby one excites at a lower wavelength and
wants to record the emitted light from the acceptor fluorophore. Therefore, having a long-pass filter separating the donor
excitation and acceptor emission wavelengths will prevent any
scattered light of the incident wavelength or FRET donor
emission from contributing to the fluorescence signal detected
by the instrument. Other filters can be used such as band-pass
filters to detect only a specific range of light depending on
experimental conditions. For a good example of this see Gzyl
and Wieden [9].
13. The xenon arc lamp needs to reach operating temperature prior
to the first measurement to achieve a stable signal (typically
15 min prior to the first measurement).
14. Each syringe holds one reaction solution, generally keeping the
two molecules of interest separate before mixing. Upon mixing, the solutions dilute each other by half if both syringes are
the identical volume, thus the final concentration of each reactant in the cuvette will be half that of the individual reaction
solutions before mixing. Therefore, it is important to prepare
reaction solutions that are double the desired reaction concentration. Some stopped-flow instruments have three or more
syringes so that three different reactants can be rapidly or
sequentially mixed without having to incubate two with each
other prior, and as such, one must be careful with the dilution
upon mixing three syringes and the desired concentrations of
each reactant in the cuvette. Additionally, the syringes can be of
different sizes having different diameters such that upon
moving the drive piston an unequal volume of solution in
either syringe is mixed resulting in a greater dilution than 1:1
which can be beneficial depending on the experiment. Protein
refolding experiments tend to utilize stopped-flow experimental setups with different syringe sizes such as in Visconti
et al. [10].
15. The incubation temperature is dependent on the particular
experiment and/or molecular system in the experiment and
thus can be adjusted to suit the respective experimental
conditions.
16. It is important to centrifuge each reaction solution at maximal
speed in a tabletop microcentrifuge before loading into the
stopped-flow to remove any precipitate. Precipitates can clog
the lines of the stopped-flow and/or interfere with subsequent
experiments if not cleaned properly. Furthermore, any precipitate that is not removed and part of a mixed sample will scatter
light during detection, thus interfering with the measurement.
284
Harland E. Brandon and Hans-Joachim Wieden