Protein Protocols Protein Protocols

308 Circolo and Gulati
weak β-emitters ( 35 S and 14 C), and permits detection of radioactivity from 3 H, virtually
undetectable with simple autoradiography. Gels are impregnated with scintillant
or fluors, which come in direct contact with the low-energy particles emitted by 35 S,
14 C, and 3 H. In response to radiation, fluors emit photons that react with the silver
halide crystals in the emulsion of the film. Because the wavelength of the emitted
photons depends on the fluorescent chemical and not on the radioactive emission of
the isotope, the same type of film can be used to detect radioactivity from isotopes of
varying energies (7).
In this chapter, we describe the most common techniques of autoradiography and
fluorography, and a double silver-staining method for acrylamide gels that approaches
the sensitivity of autoradiography and that may be used for those experiments in which
radiolabeling of proteins is not easily obtained (see Note 1). Autoradiography of wet
and dry gels is described in Subheading 3.1., and protocols for fluorography and methods
for quantification of radioactive proteins in polyacrylamide gels are given in Subheading
3.2. Double silver staining is described in Subheading 3.3.
2. Materials
2.1. Autoradiography
1. X-ray film (Kodak XR [Rochester, NY], Fuji RX [Pittsburgh, PA], Amersham [Arlington
Heights, IL], or equivalent).
2. Plastic wrap (e.g., Saran Wrap).
3. 3MM Whatman paper.
4. SDS-PAGE fixing solution: 46% (v/v) methanol, 46% (v/v) water, 8% (v/v) acetic acid
glacial. Store in an air-tight container. It is stable for months at room temperature.
2.2. Fluorography
1. X-ray blue-sensitive film (Kodak XAR-5, Amersham, or equivalent).
2. Plastic wrap.
3. 3MM Whatman paper.
4. SDS-PAGE fixing solution (as in Subheading 2.1.).
5. Coomassie blue staining solution: 0.125% Coomassie brilliant blue R250 in SDS-PAGE
fixing solution. Stir overnight to dissolve, filter through a Whatman paper, and store at
room temperature protected from light. It is stable for several weeks. For longer storage,
dissolve the Coomassie blue in 46% water and 46% methanol only, and add 8% acetic
acid glacial just before use.
6. Commercially available autoradiography enhancers (acidic acid-based: En3Hance from
Dupont [Mount Prospect, IL], water-soluble: Fluoro-Hance from RPI [Dupont NEN,
Boston, MA], or equivalent).
7. Hydrogen peroxide (15% solution).
8. Scintillation fluid.
2.3. Double Silver Staining
1. Silver stain kit (Bio-Rad, Hercules, CA).
2. Methanol, ethanol, acetic acid glacial.
3. Solution A: Sodium thiosulfate 436 g/L.
4. Solution B: Sodium chloride 37 g, cupric sulfate 37 g, ammonium hydroxide 850 mL to 1 L
of dd H2O. Store these solutions at room temperature.