be detected at the gain settings used; 100 nM fluorescein is
recommended as a default. Avoid trapping of air bubbles as
they might interfere with the detection of the signal, which can
typically lead to the loss of magnet angle or inability to lock the
magnet angle at all. Once filled, the calibration cell can be used
for several runs without changing the fluorescein solution.
Please check regularly before each run whether a bigger air
bubble has formed that might interfere with the detection of
the signal.
3. As we observed sticking of FITC-labeled clamp loader to the
material of the SEC column, we added 1 μM BSA to the
running buffer, and by this, a significantly higher yield (twoto threefold) of labeled protein was obtained.
4. It is recommended to measure absorbance spectra instead of
absorbance at single wavelengths only. Make sure that your
spectra look as expected. Absorbing contaminations and light
scattering due to aggregates might interfere with the determination of DNA and protein concentrations using UV spectroscopy. A good estimate to assess the purity and quality of DNA
and protein solutions is the absorbance at 320 nm (A320) and
the ratios of A260/A280 and A260/A230 and A260/A280,
respectively. For DNA, an A260/A280 ratio about 1.8 and an
A260/A230 ratio about 2 are expected for pure solutions. If
A260/A230 is significantly lower, it may indicate the presence
of contaminations that absorb at 230 nm, e.g., residual impurities from oligonucleotide synthesis. Such impurities can be
removed by a purification step based on silica adsorption of the
DNA. As A260/A280 might be influenced by the pH, it is
recommended to measure the absorbance of nucleic acid solutions at a slightly alkaline pH [33]. For tryptophan-containing
proteins, an A260/A280 ratio about 0.6 is expected for pure
solutions. Higher A260/A280 ratios indicate a contamination
of the protein solution with nucleotides or nucleic acids. Often,
nucleotides and nucleic acids can be removed from protein
preparations by (NH 4 ) 2 SO 4 precipitation of the protein.
High A320 values might be the result from light scattering of
aggregates, which can be removed by high-speed centrifugation or passing the solution through a sterile filter (e.g., PDVF
membrane, 0.22 μm pore size).
5. Since fluorescence is detected best in the solution directly
beneath the upper window (see Subheading 1.1), the use of
12-mm two-channel centerpieces will require an unnecessary
high sample volume (400 μL). The usage of 3 mm centerpieces
will reduce the required volume by a factor of four, but standard Beckman Coulter AUC cell housings require in this case
the placement of a 4.5-mm spacer ring between the screw ring
and the upper window assembly to ascertain that the sample
416
Andrea Bogutzki and Ute Curth
recommended as a default. Avoid trapping of air bubbles as
they might interfere with the detection of the signal, which can
typically lead to the loss of magnet angle or inability to lock the
magnet angle at all. Once filled, the calibration cell can be used
for several runs without changing the fluorescein solution.
Please check regularly before each run whether a bigger air
bubble has formed that might interfere with the detection of
the signal.
3. As we observed sticking of FITC-labeled clamp loader to the
material of the SEC column, we added 1 μM BSA to the
running buffer, and by this, a significantly higher yield (twoto threefold) of labeled protein was obtained.
4. It is recommended to measure absorbance spectra instead of
absorbance at single wavelengths only. Make sure that your
spectra look as expected. Absorbing contaminations and light
scattering due to aggregates might interfere with the determination of DNA and protein concentrations using UV spectroscopy. A good estimate to assess the purity and quality of DNA
and protein solutions is the absorbance at 320 nm (A320) and
the ratios of A260/A280 and A260/A230 and A260/A280,
respectively. For DNA, an A260/A280 ratio about 1.8 and an
A260/A230 ratio about 2 are expected for pure solutions. If
A260/A230 is significantly lower, it may indicate the presence
of contaminations that absorb at 230 nm, e.g., residual impurities from oligonucleotide synthesis. Such impurities can be
removed by a purification step based on silica adsorption of the
DNA. As A260/A280 might be influenced by the pH, it is
recommended to measure the absorbance of nucleic acid solutions at a slightly alkaline pH [33]. For tryptophan-containing
proteins, an A260/A280 ratio about 0.6 is expected for pure
solutions. Higher A260/A280 ratios indicate a contamination
of the protein solution with nucleotides or nucleic acids. Often,
nucleotides and nucleic acids can be removed from protein
preparations by (NH 4 ) 2 SO 4 precipitation of the protein.
High A320 values might be the result from light scattering of
aggregates, which can be removed by high-speed centrifugation or passing the solution through a sterile filter (e.g., PDVF
membrane, 0.22 μm pore size).
5. Since fluorescence is detected best in the solution directly
beneath the upper window (see Subheading 1.1), the use of
12-mm two-channel centerpieces will require an unnecessary
high sample volume (400 μL). The usage of 3 mm centerpieces
will reduce the required volume by a factor of four, but standard Beckman Coulter AUC cell housings require in this case
the placement of a 4.5-mm spacer ring between the screw ring
and the upper window assembly to ascertain that the sample
416
Andrea Bogutzki and Ute Curth