Proteins are denatured and disulfide bridges are broken so that the
unfolded monomers bind the negatively charged SDS homogeneously along the amino acid chain, abolishing the intrinsic
charge of the proteins and resulting in ellipsoid micelles of a constant negative charge proportional to the protein length (1.4 g SDS
are bound per 1 g protein). Thus, migration during electrophoresis
mostly depends on the proteins’ size in their unfolded state and one
protein will give just one band. In discontinuous electrophoresis
systems, the gel matrix contains two layers: a stacking gel with
neutral pH and large pores to enable the concentration of the
proteins, and a resolving/separating gel with basic pH and small
pores to separate the proteins. Subsequent staining visualizes the
proteins that have migrated as separate bands. Many staining methods are available, which differ in their sensitivity and thus their
detection limit (Subheading 2.2.1). Appropriate staining is the
most crucial aspect in detecting contaminants by gel electrophoresis. A (commercially available) size marker is usually loaded in one
lane, allowing the estimation of the molecular weight (MW) of the
proteins in the sample by comparison of positions. This molecular
weight estimate can be made more accurate by careful preparation
of the gel and subsequent quantitative analysis of the protein positions [see Note 2].
In native PAGE, the sample buffer does not contain SDS,
proteins remain in their native state, and complexes (e.g., protein:
protein or protein:nucleic acid) are not disrupted if their affinity is
high enough. The electrophoretic mobility of the proteins/protein
complexes depends on shape, charge, and molecular weight.
Hence, in addition to purity, structural homogeneity of the sample
can be assessed and the molecular weight of complexes may be
estimated via comparison with proteins/complexes of similar
shape and known molecular weight. Here, however, molecular
weight standards allow only a rough approximation due to inevitable differences in shape. As no SDS is present, buffers must be
chosen carefully to ensure that proteins/protein complexes are
charged (i.e., that the pH is away from the protein’s isoelectric
point) and will migrate during electrophoresis. Usually, in native
PAGE, both the pH and the acrylamide concentration have to be
adapted and optimized for each sample. The variant method of blue
native PAGE is often used for the analysis of membrane proteins.
Here negatively charged Coomassie Brilliant Blue dye is present in
the running buffer and binds to hydrophobic patches of the proteins, replacing the detergent without denaturing them (although
this detergent effect can itself cause the dissociation of complexes).
For reviews on the significance, methods, and protocols for native
gel analysis of protein complexes as well as membrane protein
complexes, see Refs. 9 and 10.
Protein Quality Control
7
unfolded monomers bind the negatively charged SDS homogeneously along the amino acid chain, abolishing the intrinsic
charge of the proteins and resulting in ellipsoid micelles of a constant negative charge proportional to the protein length (1.4 g SDS
are bound per 1 g protein). Thus, migration during electrophoresis
mostly depends on the proteins’ size in their unfolded state and one
protein will give just one band. In discontinuous electrophoresis
systems, the gel matrix contains two layers: a stacking gel with
neutral pH and large pores to enable the concentration of the
proteins, and a resolving/separating gel with basic pH and small
pores to separate the proteins. Subsequent staining visualizes the
proteins that have migrated as separate bands. Many staining methods are available, which differ in their sensitivity and thus their
detection limit (Subheading 2.2.1). Appropriate staining is the
most crucial aspect in detecting contaminants by gel electrophoresis. A (commercially available) size marker is usually loaded in one
lane, allowing the estimation of the molecular weight (MW) of the
proteins in the sample by comparison of positions. This molecular
weight estimate can be made more accurate by careful preparation
of the gel and subsequent quantitative analysis of the protein positions [see Note 2].
In native PAGE, the sample buffer does not contain SDS,
proteins remain in their native state, and complexes (e.g., protein:
protein or protein:nucleic acid) are not disrupted if their affinity is
high enough. The electrophoretic mobility of the proteins/protein
complexes depends on shape, charge, and molecular weight.
Hence, in addition to purity, structural homogeneity of the sample
can be assessed and the molecular weight of complexes may be
estimated via comparison with proteins/complexes of similar
shape and known molecular weight. Here, however, molecular
weight standards allow only a rough approximation due to inevitable differences in shape. As no SDS is present, buffers must be
chosen carefully to ensure that proteins/protein complexes are
charged (i.e., that the pH is away from the protein’s isoelectric
point) and will migrate during electrophoresis. Usually, in native
PAGE, both the pH and the acrylamide concentration have to be
adapted and optimized for each sample. The variant method of blue
native PAGE is often used for the analysis of membrane proteins.
Here negatively charged Coomassie Brilliant Blue dye is present in
the running buffer and binds to hydrophobic patches of the proteins, replacing the detergent without denaturing them (although
this detergent effect can itself cause the dissociation of complexes).
For reviews on the significance, methods, and protocols for native
gel analysis of protein complexes as well as membrane protein
complexes, see Refs. 9 and 10.
Protein Quality Control
7