of acrylamide used controls the size of the pores; the higher the
percentage, the smaller the pore. SDS (sodium dodecyl sulfate) is a
strong detergent with a long hydrophobic hydrocarbon tail and a
negatively charged extremity. It interacts with proteins by binding
their hydrophobic regions through its hydrocarbon part. By binding to the protein, the SDS prevents its folding. The native structure of the protein is therefore denatured, and an apparent negative
charge is then conferred to the protein. In the presence of SDS, all
the proteins will, therefore, have an apparent negative charge proportional to their polypeptide chain length. This means that only
the molecular weight of the proteins will be the factor of their
separation. Using the determined molecular mass, the presence of
a given known protein will be evaluated.
1.1.2 Capillary Gel
Electrophoresis
SDS capillary gel electrophoresis (SDS-CGE), also called capillary
gel electrophoresis (CGE), is another technique to easily determine
the purity of a PDZ construct. This technique is typically used to
separate proteins according to their size in a capillary which is filled
with polyacrylamide gel and SDS. The presence of SDS aids the
electrophoretic mobility of proteins, as it coats their surface proportional to their size. Consequently, the molecular structure will
have little influence on mobility, so macromolecules will migrate
according to their molecular mass very similarly to the SDS-PAGE
technique.
CGE has many advantages over classical SDS-PAGE, including
on-capillary detection, high separation efficiency, the capability of
accurate protein quantification and molecular weight determination, and adaptability to high-throughput method with high
reproducibility.
1.1.3 UV–Visible
Spectroscopy Between
200 nm and 340 nm
Absorption spectrophotometry is the measure of the attenuation of
the light passing through a medium in order to obtain the concentrations of absorbent substances (chromophores).
PDZs can be analyzed by absorption spectrophotometry. These
biomolecules have two main absorption band in the near-UV
region and do not absorb in the visible region (400–800 nm) of
the electromagnetic spectrum. This near-UV absorption allows to
quantify PDZs and provides information about their purity.
In the absorption spectrum of protein, a maximal absorption
band can be found around 280 nm (less intense) and between
200 and 220 nm (more intense). The bands at 280 nm and
205 nm correspond to transitions π ! π* in aromatic α-amino
acids (tyrosine, phenylalanine, and tryptophan) and in amide
groups (peptide bonds), respectively. At 280 nm, tyrosine, cystine,
and tryptophan have specific molar absorptivities that allow to
calculate a molar absorption coefficient for a given protein sequence
(http://web.expasy.org/protparam/; [12]). The main contribution at 280 nm comes from the tryptophan and tyrosine with an
PDZ Sample Quality Assessment
91
percentage, the smaller the pore. SDS (sodium dodecyl sulfate) is a
strong detergent with a long hydrophobic hydrocarbon tail and a
negatively charged extremity. It interacts with proteins by binding
their hydrophobic regions through its hydrocarbon part. By binding to the protein, the SDS prevents its folding. The native structure of the protein is therefore denatured, and an apparent negative
charge is then conferred to the protein. In the presence of SDS, all
the proteins will, therefore, have an apparent negative charge proportional to their polypeptide chain length. This means that only
the molecular weight of the proteins will be the factor of their
separation. Using the determined molecular mass, the presence of
a given known protein will be evaluated.
1.1.2 Capillary Gel
Electrophoresis
SDS capillary gel electrophoresis (SDS-CGE), also called capillary
gel electrophoresis (CGE), is another technique to easily determine
the purity of a PDZ construct. This technique is typically used to
separate proteins according to their size in a capillary which is filled
with polyacrylamide gel and SDS. The presence of SDS aids the
electrophoretic mobility of proteins, as it coats their surface proportional to their size. Consequently, the molecular structure will
have little influence on mobility, so macromolecules will migrate
according to their molecular mass very similarly to the SDS-PAGE
technique.
CGE has many advantages over classical SDS-PAGE, including
on-capillary detection, high separation efficiency, the capability of
accurate protein quantification and molecular weight determination, and adaptability to high-throughput method with high
reproducibility.
1.1.3 UV–Visible
Spectroscopy Between
200 nm and 340 nm
Absorption spectrophotometry is the measure of the attenuation of
the light passing through a medium in order to obtain the concentrations of absorbent substances (chromophores).
PDZs can be analyzed by absorption spectrophotometry. These
biomolecules have two main absorption band in the near-UV
region and do not absorb in the visible region (400–800 nm) of
the electromagnetic spectrum. This near-UV absorption allows to
quantify PDZs and provides information about their purity.
In the absorption spectrum of protein, a maximal absorption
band can be found around 280 nm (less intense) and between
200 and 220 nm (more intense). The bands at 280 nm and
205 nm correspond to transitions π ! π* in aromatic α-amino
acids (tyrosine, phenylalanine, and tryptophan) and in amide
groups (peptide bonds), respectively. At 280 nm, tyrosine, cystine,
and tryptophan have specific molar absorptivities that allow to
calculate a molar absorption coefficient for a given protein sequence
(http://web.expasy.org/protparam/; [12]). The main contribution at 280 nm comes from the tryptophan and tyrosine with an
PDZ Sample Quality Assessment
91