For running the SDS-PAGE, a tris(hydroxymethyl)aminomethane (Tris)-glycine-HCl running buffer of 25 mM Trisbase (pH 8.3), 192 mM glycin, and 0.01% (w/v) SDS is usually
used; the same buffer is used as anode and cathode buffer. Many
alternatives to the Tris-glycine system are available, which may be
better suited for a particular target protein. Of particular note, the
Tris-glycine-HCl buffer system has relatively poor separation for
proteins and peptides <15 kDa and above 200 kDa. Good separation from 5 to 30 kDa can be achieved by the method developed by
Sch€ agger and von Jagow [44], which uses a small pore gel formulation and a Tris-tricine buffer system. A Tris-acetate system can be
used to improve separation of higher molecular weight species.
Glycoproteins do not bind as many SDS molecules as nonglycosylated proteins and, consequently, migrate more slowly than nonglycosylated proteins of the same size. A Tris-borate-EDTA buffer
can be used for preparing and running such samples (replacing the
Tris-HCl) as borate will bind the sugars, giving them a negative
charge so that the migration speed is increased.
Even slight modifications of a gel electrophoresis protocol may
significantly affect the results for a certain sample. Thus, in order to
obtain high reproducibility, a standard operating procedure should
be established that is strictly followed, especially for assessing batchto-batch consistency.
Staining
The choice of the staining method depends on the required sensitivity, reproducibility, and downstream applications. For example, if
10μg of protein is loaded into a lane of the gel and contaminants of
<1% should be detected, a staining method with a sensitivity
<100 ng per band is required.
The most common method for in-gel protein staining is with
Coomassie Brilliant Blue G-250 or R-250 [45, 46] as it involves a
ready-to-use reagent that can be easily prepared in the lab, is quick,
and does not permanently chemically modify the target proteins.
Under acidic conditions, the Coomassie dye binds to basic and
hydrophobic residues while acetic acid fixes the proteins. As the
proteins are not chemically modified, excised protein bands can be
destained completely and the target proteins can be recovered for
downstream applications, such as intact mass or sequence analysis
by MS. The detection limit of Coomassie Brilliant Blue is approximately 25 ng, although in some cases amounts down to 10 ng per
band may be detected. Coomassie staining is routinely used in
many labs.
A simple protocol is presented below:
1. Wash the gel with water to remove residual SDS, which interferes with dye binding.
2. Incubate the gel in a staining solution at approximately 50
C
for 10–15 min (there are different compositions for staining
24
Bertrand Raynal et al.
used; the same buffer is used as anode and cathode buffer. Many
alternatives to the Tris-glycine system are available, which may be
better suited for a particular target protein. Of particular note, the
Tris-glycine-HCl buffer system has relatively poor separation for
proteins and peptides <15 kDa and above 200 kDa. Good separation from 5 to 30 kDa can be achieved by the method developed by
Sch€ agger and von Jagow [44], which uses a small pore gel formulation and a Tris-tricine buffer system. A Tris-acetate system can be
used to improve separation of higher molecular weight species.
Glycoproteins do not bind as many SDS molecules as nonglycosylated proteins and, consequently, migrate more slowly than nonglycosylated proteins of the same size. A Tris-borate-EDTA buffer
can be used for preparing and running such samples (replacing the
Tris-HCl) as borate will bind the sugars, giving them a negative
charge so that the migration speed is increased.
Even slight modifications of a gel electrophoresis protocol may
significantly affect the results for a certain sample. Thus, in order to
obtain high reproducibility, a standard operating procedure should
be established that is strictly followed, especially for assessing batchto-batch consistency.
Staining
The choice of the staining method depends on the required sensitivity, reproducibility, and downstream applications. For example, if
10μg of protein is loaded into a lane of the gel and contaminants of
<1% should be detected, a staining method with a sensitivity
<100 ng per band is required.
The most common method for in-gel protein staining is with
Coomassie Brilliant Blue G-250 or R-250 [45, 46] as it involves a
ready-to-use reagent that can be easily prepared in the lab, is quick,
and does not permanently chemically modify the target proteins.
Under acidic conditions, the Coomassie dye binds to basic and
hydrophobic residues while acetic acid fixes the proteins. As the
proteins are not chemically modified, excised protein bands can be
destained completely and the target proteins can be recovered for
downstream applications, such as intact mass or sequence analysis
by MS. The detection limit of Coomassie Brilliant Blue is approximately 25 ng, although in some cases amounts down to 10 ng per
band may be detected. Coomassie staining is routinely used in
many labs.
A simple protocol is presented below:
1. Wash the gel with water to remove residual SDS, which interferes with dye binding.
2. Incubate the gel in a staining solution at approximately 50
C
for 10–15 min (there are different compositions for staining
24
Bertrand Raynal et al.