Protein Protocols Protein Protocols

Autoradiography and Fluorography 311
tion of the fluors may occur, resulting in reduced sensitivity. For a longer period of storage
prior to exposure, freeze the gel at –70°C.
9. If water-soluble fluorography solutions are used (e.g., Fluoro-Hance from R.P.I. or equivalent),
the gel must be equilibrated in water after destaining, and steps 10 and 11 should be
followed.
10. Discard the destaining solution, and wash the gel in distilled water for 30 min at room
temperature, with shaking.
11. Discard the water and impregnate the gel with the enhancer for 30 min at room temperature
with shaking. Remove the enhancer, place the gel over two wet pieces of filter paper,
cover with a plastic wrap, and dry under vacuum with heat (60–80°C) for 2 h (see Note 3).
Expose the gel as described above. (Flouro-Hance can be reused, but should be discarded
as soon as the solution shows sign of discoloration) (see Notes 2–7).
If the gels are not stained with Coomassie blue, after electrophoresis, place the gel in
SDS-PAGE fixing solution, and incubate for 45 min at room temperature, with gentle
shaking. After incubation, discard the fixing solution and impregnate the gel with
enhancer as described (steps 5 or 9).
12. Radioactivity incorporated into specific proteins can be determined by cutting out the
radioactive bands from the dried fluorographed gel.
13. Precisely position the film over the gel. With a sharp blade or scalpel, cut out the area of
the gel corresponding to the band on the film. Also cut out an area of the gel free from
radioactivity immediately below (or above) the radioactive band, for subtraction of background.
14. Place each gel slice into a scintillation vial (detaching the filter paper from the slice is not
necessary), add 1 mL of a 15% solution of hydrogen peroxide, and incubate overnight in a
water bath at 60°C to digest the gel and release the radioactivity.
15. After incubation, allow the vials to cool down to room temperature, add scintillation fluid,
and measure the radioactivity in a scintillation counter. Alternatively, autoradiographic
images can be quantitated by densitometric scanning of different exposures of the film
(see Note 8).
3.3. Double Silver Staining (see Note 9)
1. After electrophoresis, transfer the gel in a glass tray containing 40% methanol and 10%
acetic acid, and incubate at room temperature for at least 30 min (longer periods of time
have no detrimental effect).
2. Discard this solution, and incubate for 15 min in 10% ethanol, 5% acetic acid.
3. Repeat step 2 one more time.
4. Add Oxidizer (diluted according to the manufacturer’s protocol) and incubate 5 min,
taking care that the gel is completely submerged in the solution.
5. Rinse the gel twice in distilled water.
6. Incubate 15 min in double-distilled water.
7. Repeat step 6 until the yellow color is completely removed from the gel.
8. Add Silver reagent (diluted according to the manufacturer’s protocol), and incubate for 15 min.
9. Wash the gel once in double-distilled water.
10. Add developer (prepared according to the manufacturer’s protocol), swirl the gel for 30 s,
discard the solution, and wash once in double-distilled water.
11. Repeat step 10 and develop the gel until bands appear and the mol-wt markers become
clearly visible.
12. Stop the reaction with 5% acetic acid, incubate 5 min, and wash with double-distilled water.
13. Add 3.5% solution A and 3.5% solution B, and incubate 5–10 min, or until the gel is clear.