Protein Protocols Protein Protocols

312 Circolo and Gulati
14. Incubate two times in 10% acetic acid, 30 min each time.
15. Restain the gel by repeating steps 1–12.
16. Dry the gel in a slab dryer with heat and vacuum, or use a gel rap (Bio-Rad or equivalent),
and dry overnight.
4. Notes
1. Autoradiography of SDS-PAGE is a powerful technique that permits detection of very
low amounts of protein. However, in some instances, radioactive protein labeling cannot
be easily accomplished. For example, metabolic labeling requires active protein synthesis
(12); thus, proteins present in body fluids or in tissue biopsy cannot be labeled (13). Moreover,
in in vivo animal experiments, it is often difficult to obtain radiolabeled proteins
with high specific activity. In this case, silver staining of gels can be used as an alternative
method, since it approaches the sensitivity of autoradiography (14). We have developed a
double silver-staining technique that is about 10-fold more sensitive than the conventional
silver staining. This method has not been previously published in detail, except for figures
presented elsewhere (15) (see Subheading 3.3.).
2. Gels of high polyacrylamide concentration (>10%) or gradient gels (acrylamide concentration
5–15%) may crack when dried. This problem is reduced by adding glycerol (1–5%)
before drying. When acid-based fluorography enhancers are used, the glycerol should be
added during the fluors precipitation step in water after removal of the enhancer. When
water-soluble enhancers are used, the glycerol is added during equilibration of the gel in
water, before addition of the enhancer. If the concentration of glycerol is too high, gels are
difficult to dry, and they may stick to the film. Addition of the enhancer does not increase
the cracking. We currently use water-based enhancers for our experiments, because they
give sharp autoradiography images and good sensitivity, and can be reused for several
gels. Fluorography with commercially available enhancers is simpler and less tedious than
the traditional method with PPO-DMSO, and the results are as good as, or better than,
those obtained with this method.
3. Enhancers are also used to increase the sensitivity of the autoradiograpy of DNA and
RNA of agarose, acrylamide, or mixed gels. When enhancers are used, the gels must be
dried at the suggested temperature, because excessive heat will cause damage of the fluors
crystal of the enhancer and formation of brown spots on the surface of the gel.
4. Cracking may also be owing to the formation of air bubbles between the gel and the rubber
cover of the dryer. This is generally caused by a weak vacuum that is insufficient to
maintain the gel well adherent to the paper filter and to the dryer’s tray. Air bubbles can be
eliminated by rolling a pipet over the rubber cover while the vacuum is being applied.
5. Also, excessive stretching of the gel during the transfer to the filter paper may contribute
to cracking, particularly for gels that contain high acrylamide concentration. A filter paper
should be placed under the gel when removing it from the solution, and the method
described in Subheading 3.1.2. should be used for larger gels.
6. It is always necessary to cover the gel with a plastic wrap to prevent sticking to the cover
of the dryer. In addition, the vacuum should never be released during the drying procedure
until the gel is completely dry, since this will cause the gel to shatter.
7. Staining of the gels with Coomassie blue before fluorography may quench the effect of
the enhancer, particularly when low amounts of radioactivity are used. Therefore, the gels
must be destained thoroughly, until the background is clear and only the protein bands are
stained.