32
L. CASTELLANOS-SERRA et al.
time can be shortened to 10 mins. During this step, soluble components from
electrophoresis (glycine, Tris) are substantially replaced by imidazole.
iii} Ready-to-use gels cast on plastic fIlms, as well as Tris-tricine, Tris-acetate and
Tris-urea gels can be stained following this protocol.
iv} Gels can be stored in MQW in sealed transparent bags or alternatively,
selected bands are excised and conserved at 4°C in MQW. The N-terminus of
proteins conserved in reverse stained gel bands for as long as three years
have been successfully sequenced (Castellanos-Serra, unpublished results).
v} Native gels can be stained by this protocol but the incorporation of SDS during staining may be inconvenient for some applications (i.e., determination
of the biological activity of the proteins after elution). Alternatively, SDS can
be suppressed from the imidazole solution; some proteins detected by
imidazole-zinc under native conditions may appear as positively white
stained bands.
Troubleshooting
i} Increasing the concentration of imidazole (i.e., 0.3 M or higher) culminates
in producing a deeper white background that may obscure less abundant
proteins.
ii} The incorporation of SDS into the imidazole solution facilitates the observation of the unstained bands due to the generation of an homogeneous white
stained background,. Although small variations in the concentration of SDS
from the recommended 0.1 % value can be tolerated without drastic effects
on the resulting staining pattern, at higher concentrations (about 0.5 %) the
precipitation of zinc imidazolate is inhibited, provoking no staining of the
gel.
iii} Prolonging the incubation in zinc 2+ causes an indiscriminate staining of the
gel surface, and no detection of the protein bands.
iv} In cases where these problems have occurred, they can be easily remedied by
initially destaining the gel and repeating the staining process: reverse stained
gels are destained by incubation in 50 mM Tris-O.3 M Glycine pH 8.0 for 10
minutes. The solution is discarded, the gel is washed with water to remove
glycine and then, it can be restained.
Advantages
This procedure has a high sensitivity, close to that of silver staining with the
advantages of being highly reproducible, simple, rapid, fully reversible and nondestructive.
2.2.2
Double Staining of Coomassie-blue (C8) Stained Gels
Procedure (Fernandez-Patron et al. 7995)
CB-stained gels are washed in MQW (two changes of water, 25 mins each, or
alternatively, overnight) to remove methanol and acetic acid. Then, the gel is
reverse stained as described in 2.2.1.
L. CASTELLANOS-SERRA et al.
time can be shortened to 10 mins. During this step, soluble components from
electrophoresis (glycine, Tris) are substantially replaced by imidazole.
iii} Ready-to-use gels cast on plastic fIlms, as well as Tris-tricine, Tris-acetate and
Tris-urea gels can be stained following this protocol.
iv} Gels can be stored in MQW in sealed transparent bags or alternatively,
selected bands are excised and conserved at 4°C in MQW. The N-terminus of
proteins conserved in reverse stained gel bands for as long as three years
have been successfully sequenced (Castellanos-Serra, unpublished results).
v} Native gels can be stained by this protocol but the incorporation of SDS during staining may be inconvenient for some applications (i.e., determination
of the biological activity of the proteins after elution). Alternatively, SDS can
be suppressed from the imidazole solution; some proteins detected by
imidazole-zinc under native conditions may appear as positively white
stained bands.
Troubleshooting
i} Increasing the concentration of imidazole (i.e., 0.3 M or higher) culminates
in producing a deeper white background that may obscure less abundant
proteins.
ii} The incorporation of SDS into the imidazole solution facilitates the observation of the unstained bands due to the generation of an homogeneous white
stained background,. Although small variations in the concentration of SDS
from the recommended 0.1 % value can be tolerated without drastic effects
on the resulting staining pattern, at higher concentrations (about 0.5 %) the
precipitation of zinc imidazolate is inhibited, provoking no staining of the
gel.
iii} Prolonging the incubation in zinc 2+ causes an indiscriminate staining of the
gel surface, and no detection of the protein bands.
iv} In cases where these problems have occurred, they can be easily remedied by
initially destaining the gel and repeating the staining process: reverse stained
gels are destained by incubation in 50 mM Tris-O.3 M Glycine pH 8.0 for 10
minutes. The solution is discarded, the gel is washed with water to remove
glycine and then, it can be restained.
Advantages
This procedure has a high sensitivity, close to that of silver staining with the
advantages of being highly reproducible, simple, rapid, fully reversible and nondestructive.
2.2.2
Double Staining of Coomassie-blue (C8) Stained Gels
Procedure (Fernandez-Patron et al. 7995)
CB-stained gels are washed in MQW (two changes of water, 25 mins each, or
alternatively, overnight) to remove methanol and acetic acid. Then, the gel is
reverse stained as described in 2.2.1.