Materials
Reagent- and analytical-grade zinc sulfate, imidazole, acetic acid, and sodium carbonate are obtained from Sigma (St. Louis, MO).
a) Equilibration solution (1X): 0.2 M imidazole, 0.1% (w/v) SDS.
b) Developer (1X): 0.3 M zinc sulfate.
c) Storage solution for reverse-stained gels (1X): 0.5 % (w/v) sodium carbonate.
All solutions are prepared as 10X concentrated stocks, stored at room temperature, and diluted (1:10) in distilled water, to yield the working concentration (1X) just before use.
Methods
1. Polyacrylamide Gel Electrophoresis
a) SDS-PAGE is carried out following the Laemmli method, whereas native PAGE is performed according to the Laemmli protocol but without SDS in the gel and electrophoresis solutions.
b) Conventional agarose gel electrophoresis is conducted in 0.8% agarose gels, using Tris-acetate, pH 8.0, as gel and running buffer.
2. Standard Reverse Staining of SDS-PAGE and Native PAGE Gels
The following reverse staining method detects proteins in standard polyacrylamide gels. All incubations are performed under continuous gentle agitation in a plastic or glass tray with a transparent bottom. The volume of the corresponding staining/storage solutions must be enough to cover the gel (typically 50 mL for one mini-gel [10 cm × 7 cm × 0.75 mm]).
a) Following electrophoresis, the gel is incubated for 15 min in the equilibration solution.
b) To develop the electropherogram, the imidazole solution is discarded and the gel soaked for 30–40 s in developer solution. Caution: This step must not be extended longer than 45 s or band overstaining and loss of the image will occur. Overstaining is prevented by pouring off the developer solution and rinsing the gel three to five times (approx 1 min) in excess water.
After completing the reverse staining procedure, the gel is ready for photographic recording. The ideal setup for this task involves placing the gel on a glass plate that is held a few centimeters above a black surface and is illuminated from the side. While SDS-PAGE is a commonly used method to separate complex protein mixtures with high resolution, there are situations where protein denaturation or disruption of macromolecular complexes should be avoided during electrophoresis. In these cases, native PAGE or agarose gel electrophoresis without SDS is employed. However, when using reverse staining, it is important to steer clear of SDS. Fortunately, two techniques have been developed to circumvent this issue.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.