Reversible Negative Staining of Protein on Electrophoresis Gels
33
Comments
i) After Reverse-staining, the gel will show
(a) typical CB-stained bands, which appear superimposed on larger transparent bands, originated by the highly sensitive negative staining of the
gel and
(b) reverse stained (negative) bands, which were previously undetected.
ii) The sensitivity is equivalent to that attained after imidazole-SDS-zinc staining on unstained gels.
iii) CB-stained gels can be Reverse-stained after prolonged storage of the gels in
sealed polyethylene bags.
iv) Sensitivity after Reverse-staining does not depend on the degree of destaining achieved on the CB stained gels. Both the extensively destained gels (until
obtaining an uncolored gel matrix) as well as the briefly destained (until
detection of the band) gels give a very high sensitivity upon Reverse-staining
(detection limit for streptokinase is 1.6 ng).
Advantages
Re-staining CB-stained gels with imidazole-SDS-zinc can successfully detect proteins that display markedly low affinity for CB as well as low abundant components that fall below the detection limit of CB staining. This procedure complements CB staining, giving a more realistic picture of the complexity of the sample
under analysis. This is due to
(a) its higher sensitivity and
(b) the generality of Reverse-staining, which is independent of the nature of the
proteins.
2.3
Protein Mobilization and Alkylation in Reverse-Stained Gels
2.3.1
Protein Mobilization Procedure (Fernandez-Patron et al. 1995)
Excise the protein bands of interest, incubate (2 x 5 mins) the gel slice in anyone
of the following solutions to complex zinc ions: (a) phosphate-buffered saline
(PBS), pH 7.4 containing 100 mM EDTA di-sodium salt or 0.3 M glycine, (b) 25
mM Tris, 0.3 M glycine, pH 8.0 (c) 1 % citric acid. (d) 25 mM Tris, 50 mM glycine
pH 8.0 containing 0.01 M DTT. After 1-2 minutes of incubation (with gentle hand
shaking), the Reverse-stained band becomes completely transparent. At the end
of the first 5 mins, renew the chelating buffer for additional 5 mins, and then
briefly wash with water.
2.3.2
Protein in-Gel Alkylation Procedure (Moritz et al. 1996b)
To perform in-gel alkylation of the protein band, first complex the zinc ions with
solution (d) 25 mM Tris, 50 mM glycine pH 8.0 containing 0.01 M DTT. After 1-2
minutes of incubation (with gentle shaking), the Reverse-stained band becomes
33
Comments
i) After Reverse-staining, the gel will show
(a) typical CB-stained bands, which appear superimposed on larger transparent bands, originated by the highly sensitive negative staining of the
gel and
(b) reverse stained (negative) bands, which were previously undetected.
ii) The sensitivity is equivalent to that attained after imidazole-SDS-zinc staining on unstained gels.
iii) CB-stained gels can be Reverse-stained after prolonged storage of the gels in
sealed polyethylene bags.
iv) Sensitivity after Reverse-staining does not depend on the degree of destaining achieved on the CB stained gels. Both the extensively destained gels (until
obtaining an uncolored gel matrix) as well as the briefly destained (until
detection of the band) gels give a very high sensitivity upon Reverse-staining
(detection limit for streptokinase is 1.6 ng).
Advantages
Re-staining CB-stained gels with imidazole-SDS-zinc can successfully detect proteins that display markedly low affinity for CB as well as low abundant components that fall below the detection limit of CB staining. This procedure complements CB staining, giving a more realistic picture of the complexity of the sample
under analysis. This is due to
(a) its higher sensitivity and
(b) the generality of Reverse-staining, which is independent of the nature of the
proteins.
2.3
Protein Mobilization and Alkylation in Reverse-Stained Gels
2.3.1
Protein Mobilization Procedure (Fernandez-Patron et al. 1995)
Excise the protein bands of interest, incubate (2 x 5 mins) the gel slice in anyone
of the following solutions to complex zinc ions: (a) phosphate-buffered saline
(PBS), pH 7.4 containing 100 mM EDTA di-sodium salt or 0.3 M glycine, (b) 25
mM Tris, 0.3 M glycine, pH 8.0 (c) 1 % citric acid. (d) 25 mM Tris, 50 mM glycine
pH 8.0 containing 0.01 M DTT. After 1-2 minutes of incubation (with gentle hand
shaking), the Reverse-stained band becomes completely transparent. At the end
of the first 5 mins, renew the chelating buffer for additional 5 mins, and then
briefly wash with water.
2.3.2
Protein in-Gel Alkylation Procedure (Moritz et al. 1996b)
To perform in-gel alkylation of the protein band, first complex the zinc ions with
solution (d) 25 mM Tris, 50 mM glycine pH 8.0 containing 0.01 M DTT. After 1-2
minutes of incubation (with gentle shaking), the Reverse-stained band becomes