(except weighing a large quantity of pure protein, which is only
practicable in rare cases) if a pure protein sample is used.
2.2 Methods
to Determine Purity,
Homogeneity,
and Oligomeric State
2.2.1 SDS-PAGE
and Staining
Gel electrophoresis equipment (apparatus, running buffers, sample
buffers, precast gels, etc.) can be obtained from many manufacturers. Protein gel electrophoresis is usually performed in vertical
systems where the gel is positioned between two glass plates with
the top part being immersed in anode buffer and the bottom in
contact with cathode buffer.
Polyacrylamide Gels
PA gels can be easily prepared in the lab, but commercially made precast
gels are recommended for QC purposes because they are much more
consistent in their behavior. Follow the manufacturer’s instructions for
their use. PA gels are produced by copolymerization of acrylamide
monomers and a crosslinker, usually N,N
0 -methylenebisacrylamide,
with the pore size being defined by the total concentration of acrylamide and the degree of consequent crosslinking. The acrylamide concentration (pore size) of the resolving gel is chosen depending on the
sizes of proteins to be resolved (usually 10–19% total acrylamide
concentration). With appropriate formulation, they can be used to
separate proteins <800 kDa; larger proteins will not enter the gel.
Use of gradient gels (increasing acrylamide concentration from top to
bottom) allows the separation and visualization of large (200 kDa) and
small (10 kDa) proteins on the same gel as the smaller pores slow down
and retain small proteins in the bottom of the gel while large proteins
are still separated at the top.
Discontinuous Denaturing
SDS-PAGE
For sample preparation, a sample buffer containing SDS is added to
the protein solution and the sample is incubated at >95
C for
some minutes in order to enhance denaturation. Typical sample
buffer (4Â concentrated): 260 mM Tris-HCl (pH 6.8), 400 mM
DTT, 8% (w/v) SDS, 0.02% (w/v) bromophenol blue, 40% (v/v)
glycerol. N.B. some proteins may precipitate or undergo degradation when heated (especially highly concentrated proteins or membrane protein) and sample preparation should be tested with and
without boiling. The reducing agent (DTT) should ensure that
each natively disulfide-bonded protein should run at the expected
molecular weight of its monomer. In order to test the presence of
disulfide bridges, samples can be prepared with and without reducing agent and should then be analyzed on the same gel. If intramolecular disulfide bridges are intact, the protein will be more compact
and the band should be shifted toward lower molecular weight as
compared to the reduced sample. If intermolecular disulfide
bridges are present, the protein will move toward higher molecular
weight.
Protein Quality Control
23
practicable in rare cases) if a pure protein sample is used.
2.2 Methods
to Determine Purity,
Homogeneity,
and Oligomeric State
2.2.1 SDS-PAGE
and Staining
Gel electrophoresis equipment (apparatus, running buffers, sample
buffers, precast gels, etc.) can be obtained from many manufacturers. Protein gel electrophoresis is usually performed in vertical
systems where the gel is positioned between two glass plates with
the top part being immersed in anode buffer and the bottom in
contact with cathode buffer.
Polyacrylamide Gels
PA gels can be easily prepared in the lab, but commercially made precast
gels are recommended for QC purposes because they are much more
consistent in their behavior. Follow the manufacturer’s instructions for
their use. PA gels are produced by copolymerization of acrylamide
monomers and a crosslinker, usually N,N
0 -methylenebisacrylamide,
with the pore size being defined by the total concentration of acrylamide and the degree of consequent crosslinking. The acrylamide concentration (pore size) of the resolving gel is chosen depending on the
sizes of proteins to be resolved (usually 10–19% total acrylamide
concentration). With appropriate formulation, they can be used to
separate proteins <800 kDa; larger proteins will not enter the gel.
Use of gradient gels (increasing acrylamide concentration from top to
bottom) allows the separation and visualization of large (200 kDa) and
small (10 kDa) proteins on the same gel as the smaller pores slow down
and retain small proteins in the bottom of the gel while large proteins
are still separated at the top.
Discontinuous Denaturing
SDS-PAGE
For sample preparation, a sample buffer containing SDS is added to
the protein solution and the sample is incubated at >95
C for
some minutes in order to enhance denaturation. Typical sample
buffer (4Â concentrated): 260 mM Tris-HCl (pH 6.8), 400 mM
DTT, 8% (w/v) SDS, 0.02% (w/v) bromophenol blue, 40% (v/v)
glycerol. N.B. some proteins may precipitate or undergo degradation when heated (especially highly concentrated proteins or membrane protein) and sample preparation should be tested with and
without boiling. The reducing agent (DTT) should ensure that
each natively disulfide-bonded protein should run at the expected
molecular weight of its monomer. In order to test the presence of
disulfide bridges, samples can be prepared with and without reducing agent and should then be analyzed on the same gel. If intramolecular disulfide bridges are intact, the protein will be more compact
and the band should be shifted toward lower molecular weight as
compared to the reduced sample. If intermolecular disulfide
bridges are present, the protein will move toward higher molecular
weight.
Protein Quality Control
23