solutions, a typical one is 0.025% (w/v) Coomassie Brilliant
Blue G-250 in 10% (v/v) acetic acid; ethanol or methanol may
be added in some formulations).
3. Incubate the gel in a destaining solution at approximately
50
C for 10–15 min to wash out non-bound dye (again,
there are different compositions for destaining solutions
according to the manufacturer, e.g., 10% (v/v) acetic acid;
ethanol/methanol may be added).
4. Incubate the gel in water overnight, where, optionally, a piece
of paper tissue may be added to fully wash out non-bound dye.
Usually, larger proteins tend to bind more dye than small ones.
Consequently, the intensity of bands can only be compared in terms
of weight concentrations (mg/mL) and not in terms of molar
concentrations. If quantitation by densitometric analysis is
required, a comparison with samples of the same protein at
known concentration is necessary; as well as even staining throughout the gel.
Staining with the Coomassie Brilliant Blue dye is the most
common method and is usually sufficient when working with purified recombinant proteins. If there is sufficient protein available,
but protein concentration is simply low, the sample can be concentrated by trichloroacetic acid (TCA), TCA/deoxycholate, or
TCA/acetone precipitation in order to allow more total protein
to be loaded. However, if available protein is low, it should be noted
that Coomassie staining is at least 10 times less sensitive than silver
staining, reverse zinc staining, visualization by fluorescent dyes, or
radioactive staining (rarely used in QC). The higher sensitivity
stains should also be considered whenever the main objective of
the gel electrophoretic run is the detection of impurities in the
sample. There are also stain-free techniques available, where proteins can be visualized without the need of a stain. See Note 8 for
further details on other visualization approaches.
2.2.2 Size-Exclusion
Chromatography: Static
Light Scattering
(SEC-LALS/SEC-MALS)
An appropriate chromatography column is required. Depending on
the manufacturer and the chemistry of the matrix, each column will
be specified in terms of a particular range of molecular sizes that it
can effectively separate. This is normally given in terms of the
molecular mass of globular proteins. One should keep in mind
that the molecular mass is not identical to the hydrodynamic size,
and for nonglobular proteins, substantial adjustment may be
required (i.e., an elongated 60-kDa protein may behave like a
160-kDa globular protein). In practice, the column should be
chosen for its ability to elute the protein of interest at a volume
toward the center of the upper and lower limit, and in particular
that the target protein elution is well-separated from the void
volume so that aggregates are removed. For quantitative, analytical
Protein Quality Control
25
Blue G-250 in 10% (v/v) acetic acid; ethanol or methanol may
be added in some formulations).
3. Incubate the gel in a destaining solution at approximately
50
C for 10–15 min to wash out non-bound dye (again,
there are different compositions for destaining solutions
according to the manufacturer, e.g., 10% (v/v) acetic acid;
ethanol/methanol may be added).
4. Incubate the gel in water overnight, where, optionally, a piece
of paper tissue may be added to fully wash out non-bound dye.
Usually, larger proteins tend to bind more dye than small ones.
Consequently, the intensity of bands can only be compared in terms
of weight concentrations (mg/mL) and not in terms of molar
concentrations. If quantitation by densitometric analysis is
required, a comparison with samples of the same protein at
known concentration is necessary; as well as even staining throughout the gel.
Staining with the Coomassie Brilliant Blue dye is the most
common method and is usually sufficient when working with purified recombinant proteins. If there is sufficient protein available,
but protein concentration is simply low, the sample can be concentrated by trichloroacetic acid (TCA), TCA/deoxycholate, or
TCA/acetone precipitation in order to allow more total protein
to be loaded. However, if available protein is low, it should be noted
that Coomassie staining is at least 10 times less sensitive than silver
staining, reverse zinc staining, visualization by fluorescent dyes, or
radioactive staining (rarely used in QC). The higher sensitivity
stains should also be considered whenever the main objective of
the gel electrophoretic run is the detection of impurities in the
sample. There are also stain-free techniques available, where proteins can be visualized without the need of a stain. See Note 8 for
further details on other visualization approaches.
2.2.2 Size-Exclusion
Chromatography: Static
Light Scattering
(SEC-LALS/SEC-MALS)
An appropriate chromatography column is required. Depending on
the manufacturer and the chemistry of the matrix, each column will
be specified in terms of a particular range of molecular sizes that it
can effectively separate. This is normally given in terms of the
molecular mass of globular proteins. One should keep in mind
that the molecular mass is not identical to the hydrodynamic size,
and for nonglobular proteins, substantial adjustment may be
required (i.e., an elongated 60-kDa protein may behave like a
160-kDa globular protein). In practice, the column should be
chosen for its ability to elute the protein of interest at a volume
toward the center of the upper and lower limit, and in particular
that the target protein elution is well-separated from the void
volume so that aggregates are removed. For quantitative, analytical
Protein Quality Control
25