A simple protocol to run an NMR experiment is given below:
1. Transfer each sample into an NMR tube, add 1–5% D 2 O and
insert the sample plus the spinner into the magnet with the
sampler changer.
2. Adjust manually the probe equipped with an ATM module for
each nucleus adapted to the experience to ensure the best
sensitivity of the probe.
3. Lock the magnet to keep its field constant and stable during the
data acquisition and shim the magnet to make it uniform and
homogenous for a sample to get a reasonable resolution and
line shape.
4. Determine the 90
transmitter pulse.
5. Setup Parameters and Acquire Data.
6. Process Data to convert data into NMR spectra.
7. Conformational stability/folding state.
4 Notes
1. Absorbance at the characteristic wavelength of 280 nm (i.e.,
A280 nm) cannot be used for quantitative analysis without
protein sequence since the amino acid composition varies
from one protein to another. Indeed, variations in amino acid
composition give molar absorptivities (ε280 nm) that vary
from one protein to another.
2. Add glycerol gives the solution better to put down samples in
the wells. If the solution is yellow while it should be blue
because BBP turns yellow at acidic pH, add a few microliters
of NaOH to turn blue the solution.
3. The materials are already fully described in [39].
4. The buffer should contain at least 50 mM of salt to avoid
interactions with the column matrix and 0.1% (w/v) NaN3 to
prevent microbial growth. All solutions must be freshly filtered
and degassed to avoid background scattering and an unstable
baseline.
5. Cuvettes with pathlengths ranging from 0.01 to 10 mm are
used with a 20-1000 μl volume of protein solution at a concentration of 50 μg/ml to 1 mg/ml.
6. If you load 10 μg that corresponds to 10 μl at 1 mg/ml of
protein, you will need sensitivity of about 100 ng per band to
detect contamination. The detection limit of Coomassie blue
staining is approximately 100 ng per band, whereas silver stains
and fluorescent dye have a detection limit of approximately
1 ng of protein per band [20].
PDZ Sample Quality Assessment
119
1. Transfer each sample into an NMR tube, add 1–5% D 2 O and
insert the sample plus the spinner into the magnet with the
sampler changer.
2. Adjust manually the probe equipped with an ATM module for
each nucleus adapted to the experience to ensure the best
sensitivity of the probe.
3. Lock the magnet to keep its field constant and stable during the
data acquisition and shim the magnet to make it uniform and
homogenous for a sample to get a reasonable resolution and
line shape.
4. Determine the 90
transmitter pulse.
5. Setup Parameters and Acquire Data.
6. Process Data to convert data into NMR spectra.
7. Conformational stability/folding state.
4 Notes
1. Absorbance at the characteristic wavelength of 280 nm (i.e.,
A280 nm) cannot be used for quantitative analysis without
protein sequence since the amino acid composition varies
from one protein to another. Indeed, variations in amino acid
composition give molar absorptivities (ε280 nm) that vary
from one protein to another.
2. Add glycerol gives the solution better to put down samples in
the wells. If the solution is yellow while it should be blue
because BBP turns yellow at acidic pH, add a few microliters
of NaOH to turn blue the solution.
3. The materials are already fully described in [39].
4. The buffer should contain at least 50 mM of salt to avoid
interactions with the column matrix and 0.1% (w/v) NaN3 to
prevent microbial growth. All solutions must be freshly filtered
and degassed to avoid background scattering and an unstable
baseline.
5. Cuvettes with pathlengths ranging from 0.01 to 10 mm are
used with a 20-1000 μl volume of protein solution at a concentration of 50 μg/ml to 1 mg/ml.
6. If you load 10 μg that corresponds to 10 μl at 1 mg/ml of
protein, you will need sensitivity of about 100 ng per band to
detect contamination. The detection limit of Coomassie blue
staining is approximately 100 ng per band, whereas silver stains
and fluorescent dye have a detection limit of approximately
1 ng of protein per band [20].
PDZ Sample Quality Assessment
119