Protein Protocols Protein Protocols

310 Circolo and Gulati
4. Flip over the plate and maintain it exactly over the tray of a gel dryer. Carefully begin to
detach the gel from one corner of the glass plate. The gel will remain adherent to the
Whatman paper and will detach easily from the plate. Carefully cover the gel with plastic
wrap, avoiding folding, and then dry the gel in a slab gel vacuum dryer for 1 h at 80°C (see
Notes 2–6).
5. When the gel is dry, remove the plastic wrap and expose to X-ray film. After an appropriate
length of time, develop the film as described in Subheading 3.1.1., step 3.
To increase the resolution of the radiogaphy, gels in which several bands of similar
molecular mass are visualized, should be exposed at room temperature, without an
intensifying screen. The use of a screen will result in increased intensity of the radioactive
signal, but in decreased sharpness of the image. SDS-PAGE or other gels in
which fewer bands need to be resolved may be exposed with an intensifying screen.
3.2. Fluorography
Gels containing weak β-emitters ( 35 S, 14 C, and 3 H) should be fixed, impregnated
with autoradiography enhancers, and dried to reduce the film exposure time necessary
for visualization of radioactive bands. SDS-PAGE should also be stained if nonradiolabeled
mol-wt markers are used and for quantitative experiments (e.g., immunoprecipitation,
detection of cell-free translated products, and so forth) where the radioactivity
contained in specific proteins is to be measured in bands cut out from the gel (8,9).
1. Turn off the power supply, remove the gel from the mold, cut a corner for orientation, and
place the gel on a tray containing Coomassie blue-staining solution (the volume of the
solution should always be adequate to cover the gel, so that it can float freely). Incubate
for 45 min at room temperature with gentle shaking.
2. Remove the staining solution, and replace with SDS-PAGE fixing solution (the staining
solution can be filtered through filter paper and reused as long as the radioactivity on it
remains low, or until the color changes from blue to purple).
3. Incubate overnight at room temperature with gentle shaking, and replace the fixing solution
at least once to accelerate the destaining. Gels should be destained until the mol-wt
markers are clearly visible and the background is clear (see Note 7).
4. Discard the fixing solution in accordance with radioactive liquid waste disposal procedures,
and add the autoradiography enhancer.
5. If the enhancer used is based on acetic acid (e.g., En 3 Hance from Dupont or its equivalent),
the gel can be soaked in the enhancer without rinsing, and steps 6–8 should be
followed (see Note 2).
6. Allow the gel to impregnate with enhancer for 1 h with gentle shaking. Initially, a white
precipitate may form on the surface of the gel, but it will disappear within the first 15 min
of impregnation. Following impregnation, discard the used enhancer solution (do not mix
with waste containing NaOH, NaHCO 3, and so forth). Add cold tap water to the gel to
precipitate the fluorescent material and incubate the gel in water for 30 min. At this stage,
the gel should appear uniformly opaque.
7. After the precipitation step, carefully place the gel over two pieces of wet filter paper, cover
with plastic wrap, and dry under heat (60 –70°C) and vacuum for 1–2 h on a slab gel dryer.
8. Remove the plastic wrap, tape the gel on a rigid support, and place it against a suitable bluesensitive
X-ray film, with an intensifying screen. Expose at –70°C for an appropriate length
of time. Do not store the gel at room temperature for >48 h before exposure, since evapora-