Reversible Negative Staining of Protein on Electrophoresis Gels
2
Procedures
2.1
Methods
31
Reagents are analytical or electrophoresis grade from international suppliers
(Merck, BioRad, Pharmacia). Solutions are prepared with high quality water (MilliQ water, MQW). Volumes of staining solutions for mini-gels (about 8 x 10 cm) are
- 100 mL; for medium-size gels (16 x 18 cm), the volume should be - 250 mL.
2.2.
Protein detection on polyacrylamide gels by Reverse-staining
2.2.1
Detection on SDS gels
Procedure (Fernandez-Patron, and Castellanos-Serra, 7990; Fernandez-Patron et
01.7992, Ortiz et 01 7992)
Prepare lO x stock staining solutions: (a) 2 M ImH, 1 % SDS and (b) 2 M zinc
sulfate. For use, dilute each stock solution 1:10 with MQW.
Imidazole step: After electrophoresis, place the gel in a transparent plastic tray,
soak for 30 s in MWQ. Incubate the gel for 15 mins in 0.2 M ImH, containing
0.1 % SDS, while shaking continuously.
Zinc step: Discard the imidazole solution. Hold the tray a few centimeters over
any dark surface to facilitate the visualization of the staining pattern, then add
0.2 M zinc sulfate while manually shaking. The gel will stain white due to the precipitation of zinc-imidazolate-SDS complex on the gel surface, leaving transparent, colorless (unstained) protein bands. When an adequate staining pattern is
attained (after 30-45 s), discard the zinc solution and rapidly rinse the gel 3-4
times in abundant MQW, to avoid overstaining.
Digital Recording of Reverse Stained gels: Place the gel on the scanner screen
and cover it with a black cardboard. Alternatively, scan the gel in a dark room
while leaving the scanned lid opened. Adjust the image for optimal contrast.
Scanning of Reverse-stained gels can also be obtained by using laser-based dens ito meters as is performed for gels stained with other positive staining methods
(Molecular Dynamics, U.S.A.).
Photography: place the gel on a glass plate, held some centimeters above a black
surface, while illuminating with fluorescent light.
Comments
i) The stock staining solutions are stable in amber bottles at room temperature
for years.
ii) Recommended incubation time in the imidazole solution is 15 mins for 10 %,
12.5 % and 15 % polyacrylamide gels. For 5 %-7.5 % polyacrylamide gels, this
2
Procedures
2.1
Methods
31
Reagents are analytical or electrophoresis grade from international suppliers
(Merck, BioRad, Pharmacia). Solutions are prepared with high quality water (MilliQ water, MQW). Volumes of staining solutions for mini-gels (about 8 x 10 cm) are
- 100 mL; for medium-size gels (16 x 18 cm), the volume should be - 250 mL.
2.2.
Protein detection on polyacrylamide gels by Reverse-staining
2.2.1
Detection on SDS gels
Procedure (Fernandez-Patron, and Castellanos-Serra, 7990; Fernandez-Patron et
01.7992, Ortiz et 01 7992)
Prepare lO x stock staining solutions: (a) 2 M ImH, 1 % SDS and (b) 2 M zinc
sulfate. For use, dilute each stock solution 1:10 with MQW.
Imidazole step: After electrophoresis, place the gel in a transparent plastic tray,
soak for 30 s in MWQ. Incubate the gel for 15 mins in 0.2 M ImH, containing
0.1 % SDS, while shaking continuously.
Zinc step: Discard the imidazole solution. Hold the tray a few centimeters over
any dark surface to facilitate the visualization of the staining pattern, then add
0.2 M zinc sulfate while manually shaking. The gel will stain white due to the precipitation of zinc-imidazolate-SDS complex on the gel surface, leaving transparent, colorless (unstained) protein bands. When an adequate staining pattern is
attained (after 30-45 s), discard the zinc solution and rapidly rinse the gel 3-4
times in abundant MQW, to avoid overstaining.
Digital Recording of Reverse Stained gels: Place the gel on the scanner screen
and cover it with a black cardboard. Alternatively, scan the gel in a dark room
while leaving the scanned lid opened. Adjust the image for optimal contrast.
Scanning of Reverse-stained gels can also be obtained by using laser-based dens ito meters as is performed for gels stained with other positive staining methods
(Molecular Dynamics, U.S.A.).
Photography: place the gel on a glass plate, held some centimeters above a black
surface, while illuminating with fluorescent light.
Comments
i) The stock staining solutions are stable in amber bottles at room temperature
for years.
ii) Recommended incubation time in the imidazole solution is 15 mins for 10 %,
12.5 % and 15 % polyacrylamide gels. For 5 %-7.5 % polyacrylamide gels, this