UV-visible spectroscopy is also useful for the detection of
nonprotein contaminants by monitoring the absorbance over a
large range (at least 240–340 nm). Contaminating nucleic acids,
reducing agents and detergents may show extra absorbance around
260 nm. The contribution of these contaminants is evaluated with
the ratio A 260nm /A 280nm , which should be lower than 0.6 [14]. We
already observed such contaminations with the first PDZ domain of
Whirlin, a protein involved in the human auditory system, which
presents a cationic patch at the surface of its PDZ1 domain. This
basic cluster of three or four Arg or Lys close to the carboxylate
binding site is common to a subset of PDZ domains that bind lipids
[40]. More generally, we might presume that PDZ domains known
to interact with lipids [41] may also interact with nucleic acids
through the cluster of basic residues. In line with this, samples
with PDZ domains having high isoelectric point (pI) values are
prone to nucleic acids contamination.
If the sample shows no sign of contamination or aggregation,
the sample concentration can be calculated using A 280nm , the protein molar absorption coefficient at the working wavelength and the
Beer–Lambert’s law [42]. However, PDZ domains are small
domains and some of them lack of tryptophan. In that case, a
weak A 280nm is obtained and might alter the precision of the
concentration. In that case, an alternative way is to use A 205nm to
determine concentration [13].
The protocol for UV spectrum measurement comprises a run
of the baseline/blank with buffer and ones with the protein solution. The exact same buffer that was used to prepare the protein
solution should be used, ideally the buffer from the last step of
purification, that is, the exclusion chromatography in the case of
MAST2-PDZ. For concentration measurement, the absorbance at
the wavelength used must be below the specified saturation range
of the instrument to keep the linear relationship between the
absorbance and the concentration. If the absorbance is too high,
the sample must be diluted and the measurement should be
repeated. PDZ spectra are also checked for aggregation around
320–340 nm and for contamination by nucleic acids at 260 nm,
for example. If any signs of aggregation or contaminants are
detected, the A 280nm and the corresponding extinction coefficient
are used to calculate the concentration of the protein in the cuvette.
A simple protocol to run a UV-visible spectrum is given below:
1. Turn on the UV-Vis spectrometer and allow the lights to warm
up for an appropriate period of time (approximately 20 min) to
stabilize.
2. Fill a cuvette with the buffer used to generate the protein
sample and make sure the outside is clean. This will serve as a
blank and help account for slight losses due to diffusion or
absorption by the solvent.
PDZ Sample Quality Assessment
109
nonprotein contaminants by monitoring the absorbance over a
large range (at least 240–340 nm). Contaminating nucleic acids,
reducing agents and detergents may show extra absorbance around
260 nm. The contribution of these contaminants is evaluated with
the ratio A 260nm /A 280nm , which should be lower than 0.6 [14]. We
already observed such contaminations with the first PDZ domain of
Whirlin, a protein involved in the human auditory system, which
presents a cationic patch at the surface of its PDZ1 domain. This
basic cluster of three or four Arg or Lys close to the carboxylate
binding site is common to a subset of PDZ domains that bind lipids
[40]. More generally, we might presume that PDZ domains known
to interact with lipids [41] may also interact with nucleic acids
through the cluster of basic residues. In line with this, samples
with PDZ domains having high isoelectric point (pI) values are
prone to nucleic acids contamination.
If the sample shows no sign of contamination or aggregation,
the sample concentration can be calculated using A 280nm , the protein molar absorption coefficient at the working wavelength and the
Beer–Lambert’s law [42]. However, PDZ domains are small
domains and some of them lack of tryptophan. In that case, a
weak A 280nm is obtained and might alter the precision of the
concentration. In that case, an alternative way is to use A 205nm to
determine concentration [13].
The protocol for UV spectrum measurement comprises a run
of the baseline/blank with buffer and ones with the protein solution. The exact same buffer that was used to prepare the protein
solution should be used, ideally the buffer from the last step of
purification, that is, the exclusion chromatography in the case of
MAST2-PDZ. For concentration measurement, the absorbance at
the wavelength used must be below the specified saturation range
of the instrument to keep the linear relationship between the
absorbance and the concentration. If the absorbance is too high,
the sample must be diluted and the measurement should be
repeated. PDZ spectra are also checked for aggregation around
320–340 nm and for contamination by nucleic acids at 260 nm,
for example. If any signs of aggregation or contaminants are
detected, the A 280nm and the corresponding extinction coefficient
are used to calculate the concentration of the protein in the cuvette.
A simple protocol to run a UV-visible spectrum is given below:
1. Turn on the UV-Vis spectrometer and allow the lights to warm
up for an appropriate period of time (approximately 20 min) to
stabilize.
2. Fill a cuvette with the buffer used to generate the protein
sample and make sure the outside is clean. This will serve as a
blank and help account for slight losses due to diffusion or
absorption by the solvent.
PDZ Sample Quality Assessment
109