3. Place the cuvette in the spectrometer (see Note 9). Make sure
to align it correctly.
4. Read for the blank assay in the range of 200–500 nm for
example for a protein sample (see Note 10). Absorbance should
be minimal, but any absorption should be deducted from
future samples. Most instruments store empty data and perform subtraction automatically.
5. Fill the cuvette with the sample. To ensure that the transfer is
quantitative, rinse the cuvette 3 times with water, then 3 times
with alcohol and dry with pressured air. Make sure the outside
is clean of fingerprints by cleaning the cuvette with a
special wipe.
6. Place the cell in the spectrometer in the correct direction. Close
the cap to avoid any ambient light and collect an absorbance
spectrum in the range of 200–500 nm.
3.2 PDZ Mass
Integrity by
MALDI-TOF
There are many ways to prepare deposits on the MALDI target with
variations in terms of the matrix concentration, the solvent used, or
the crystallization process. The purpose of the droplet preparation
on the MALDI plate is to allow the protein–matrix mixture to be
close enough to cocrystallize. The dried droplet method is the most
commonly used technique because it is swift and straightforward to
implement. There are several alternatives to proceed that are all
roughly equivalent. Indeed, it is possible to directly mix the PDZ
domain with the matrix on the MALDI plate or to premix it in a
tube and deposit the mixture on the target.
3.2.1 Total Mass
Measurement
For total mass measurement of PDZ domain (Fig. 10), the protocol is as follow:
1. Use the dried droplet method: Apply 1 μl of PDZ sample on
the plate and overlay with 1 μl of HCCA. For a PDZ domain of
15 kDa, 1 μl at 0.1 mg/ml is sufficient. For a 30 kDa PDZ
construct (a PDZ tandem for example), a concentration of
0.3 mg/ml is recommended. These concentrations are acceptable for protein samples in PBS-type buffer. For complex buffer
or low PDZ quantity see Note 11.
2. Let the cocrystals air-dry for 15 min.
3. Acquire the data in linear and positive mode on the MALDITOF mass spectrometer. Use the minimum laser power
required to get a nice spectrum (see Note 12).
4. Calibrate the experiment with a standard mixture, for instance,
the Protein Standard I from BRUKER company.
5. Analyze the data (see Note 13).
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Ce ´ lia Caillet-Saguy et al.
to align it correctly.
4. Read for the blank assay in the range of 200–500 nm for
example for a protein sample (see Note 10). Absorbance should
be minimal, but any absorption should be deducted from
future samples. Most instruments store empty data and perform subtraction automatically.
5. Fill the cuvette with the sample. To ensure that the transfer is
quantitative, rinse the cuvette 3 times with water, then 3 times
with alcohol and dry with pressured air. Make sure the outside
is clean of fingerprints by cleaning the cuvette with a
special wipe.
6. Place the cell in the spectrometer in the correct direction. Close
the cap to avoid any ambient light and collect an absorbance
spectrum in the range of 200–500 nm.
3.2 PDZ Mass
Integrity by
MALDI-TOF
There are many ways to prepare deposits on the MALDI target with
variations in terms of the matrix concentration, the solvent used, or
the crystallization process. The purpose of the droplet preparation
on the MALDI plate is to allow the protein–matrix mixture to be
close enough to cocrystallize. The dried droplet method is the most
commonly used technique because it is swift and straightforward to
implement. There are several alternatives to proceed that are all
roughly equivalent. Indeed, it is possible to directly mix the PDZ
domain with the matrix on the MALDI plate or to premix it in a
tube and deposit the mixture on the target.
3.2.1 Total Mass
Measurement
For total mass measurement of PDZ domain (Fig. 10), the protocol is as follow:
1. Use the dried droplet method: Apply 1 μl of PDZ sample on
the plate and overlay with 1 μl of HCCA. For a PDZ domain of
15 kDa, 1 μl at 0.1 mg/ml is sufficient. For a 30 kDa PDZ
construct (a PDZ tandem for example), a concentration of
0.3 mg/ml is recommended. These concentrations are acceptable for protein samples in PBS-type buffer. For complex buffer
or low PDZ quantity see Note 11.
2. Let the cocrystals air-dry for 15 min.
3. Acquire the data in linear and positive mode on the MALDITOF mass spectrometer. Use the minimum laser power
required to get a nice spectrum (see Note 12).
4. Calibrate the experiment with a standard mixture, for instance,
the Protein Standard I from BRUKER company.
5. Analyze the data (see Note 13).
110
Ce ´ lia Caillet-Saguy et al.