2. Dispense 5.6 μl/well of HT Protein 200 Sample buffer in as
many wells of a 384 well plate as required.
3. Add 4 μl/well of the PDZ dilutions to each well.
4. Boil the samples at 95
C for 5 min in a dry bath.
5. Add 20.6 μl/well of distilled H 2 O.
6. PDZ samples within the 384 well plate is then analyzed with
the High Sensitivity HT Protein Express protocol
(10–100 kDa program) with the LabChip GXII device (PerkinElmer) following supplier’s instructions. This technology
allows to electrophoretically separate, stain, destain, detect by
laser-induced fluorescence and analyze the protein samples.
The data analysis provides protein concentration, molecular
weight sizing, and purity evaluation using ladder and marker
calibration standards. With sample acquisition time of about
40 s, the instrument takes approximately 4 h to analyze
384 protein samples.
7. The LabChip GX software is then used for data analysis. This
allows to visualize results via an electropherogram or virtual gel
view (Fig. 8) or in a tabular form to export into a spreadsheet
format. At the end of the run, the concentrations of the initial
cultures of the soluble PDZ are calculated from the concentrations determined per PDZ in each serial dilution. The PDZ
lysate concentrations range from 10 μM for the least concentrated and up to 100 μM or higher. The concentration of
lysozyme is constant and used as an internal reference for
quantification. A limitation to the quantification is reached
when the construct is poorly expressed and falls in the background of the Escherichia coli proteins in the case of a lysate. On
the contrary, the electropherogram allows to easily detect contaminants (Fig. 8). This CGE approach is fast, efficient and
allows high resolving separations with low solvent consumption and minimal operating cost considering the high number
of samples. However, the LabChip GXII system is not common
in most laboratories. Its running cost, the short-use warranty of
ships and kits, can constitute a limitation. During the 4 h run, a
diminution in data quality may happen due to samples drying
into the 384 well plate.
3.1.3 UV Spectroscopy
UV spectroscopy between 220–240 nm and 340 nm is a good
quality test to determine protein concentration (using A 280nm )
and to detect aggregation and molecule contamination. Indeed,
UV-visible spectroscopy can detect the presence of large particles
such as aggregates (radius higher than 200 nm) in a protein preparation by monitoring the absorbance signal above 320 nm, where
aggregate-free protein samples are not supposed to absorb light. If
the signal increases as the wavelength diminishes between 340 nm
PDZ Sample Quality Assessment
107
many wells of a 384 well plate as required.
3. Add 4 μl/well of the PDZ dilutions to each well.
4. Boil the samples at 95
C for 5 min in a dry bath.
5. Add 20.6 μl/well of distilled H 2 O.
6. PDZ samples within the 384 well plate is then analyzed with
the High Sensitivity HT Protein Express protocol
(10–100 kDa program) with the LabChip GXII device (PerkinElmer) following supplier’s instructions. This technology
allows to electrophoretically separate, stain, destain, detect by
laser-induced fluorescence and analyze the protein samples.
The data analysis provides protein concentration, molecular
weight sizing, and purity evaluation using ladder and marker
calibration standards. With sample acquisition time of about
40 s, the instrument takes approximately 4 h to analyze
384 protein samples.
7. The LabChip GX software is then used for data analysis. This
allows to visualize results via an electropherogram or virtual gel
view (Fig. 8) or in a tabular form to export into a spreadsheet
format. At the end of the run, the concentrations of the initial
cultures of the soluble PDZ are calculated from the concentrations determined per PDZ in each serial dilution. The PDZ
lysate concentrations range from 10 μM for the least concentrated and up to 100 μM or higher. The concentration of
lysozyme is constant and used as an internal reference for
quantification. A limitation to the quantification is reached
when the construct is poorly expressed and falls in the background of the Escherichia coli proteins in the case of a lysate. On
the contrary, the electropherogram allows to easily detect contaminants (Fig. 8). This CGE approach is fast, efficient and
allows high resolving separations with low solvent consumption and minimal operating cost considering the high number
of samples. However, the LabChip GXII system is not common
in most laboratories. Its running cost, the short-use warranty of
ships and kits, can constitute a limitation. During the 4 h run, a
diminution in data quality may happen due to samples drying
into the 384 well plate.
3.1.3 UV Spectroscopy
UV spectroscopy between 220–240 nm and 340 nm is a good
quality test to determine protein concentration (using A 280nm )
and to detect aggregation and molecule contamination. Indeed,
UV-visible spectroscopy can detect the presence of large particles
such as aggregates (radius higher than 200 nm) in a protein preparation by monitoring the absorbance signal above 320 nm, where
aggregate-free protein samples are not supposed to absorb light. If
the signal increases as the wavelength diminishes between 340 nm
PDZ Sample Quality Assessment
107