Autoradiography and fluorography

4.10 Autoradiography and fluorography

The utility and sensitivity of SDS-PAGE gels can be greatly increased by the use of radioactively labelled molecules, particularly in combination with immunoprecipitation. It is also possible to convert this essentially qualitative technique into a quantitative one, as explained in the Technical notes below.

4.10.1 Direct autoradiography

MATERIALS AND EQUIPMENT SDS-PAGE gel, containing radioactively labelled proteins (see Appendix B.2.1) X-ray film Polyacrylamide gel drier

3 MM paper Cling film or Saran wrap Intensifying screen Metal X-ray cassette

METHOD

1 Remove the polyacrylamide gel from the electrophoresis apparatus and cut away the stacking gel with a scalpel blade.

2 Fix the gel for 4 h in an aqueous solution of 40% v/v methanol and 10% v/v acetic acid and then soak for 15 min in a 5% solution of acetic acid containing 0.1% glycerol.

Continued

C H A P T E R 4: Antibodies as probes

3 Lay the gel on a sheet of water-saturated 3 MM paper just larger than the gel and cover with a sheet of cling film. Take care to remove all excess water and air bubbles before trimming away the excess cling film with a scalpel blade.

4 Seal the gel into a commercial drier, preferably connected to a high-efficiency oil vacuum pump via two solid CO 2 /acetone water traps. A water vacuum pump will do, provided the water pressure is high enough.

5 Turn on the vacuum and heating and dry the gel completely. This may be determined by the change in profile of the gel or when the gel no longer feels cool to the touch, in each case as judged through the rubber sheet of the drier. Do not release the vacuum until the gel is hot.

6 In the dark room, load a metal X-ray cassette with an intensifying screen (if required; see Technical notes), film and the gel (face down on the film).

7 If an intensifying screen has been used put the cassette at –70°C, otherwise leave it at room temperature away from direct sunlight, volatile chemicals and any external source of penetrating radiation.

8 Develop the X-ray film according to the manufacturer’s instructions. The length of exposure will vary according to the type and amount of radioactivity and should be determined empirically. In some gels, it might be necessary to have both a long and short exposure to gain maximum information; for example, see Fig. 3.15.

9 Blackening of the film in areas corresponding to the protein tracks may be used to infer the presence of radioactive material in the original gel.

TECHNICAL NOTES • Direct autoradiography is only possible with the penetrating radiations emanating from 131 I,

125 I, 32 P, etc., Weaker emitters 14 C, 35 S, 3 H, etc., require fluorography: see below. • The sensitivity of detection may be increased by the use of:

(a) Intensifying screens. These are plastic sheets impregnated with heavy metal ion phosphor crystals and are used to trap radiations which pass through the X-ray film. As a consequence, they emit photons and so blacken the X-ray film on the side away from the polyacrylamide gel. In principle, it is possible to use two intensifying screens, one on either side of the film. In practice, however, this can blur the final image because of the greater distance travelled by the radioactive and photon radiations.

(b) Preflashed film. X-ray film is relatively insensitive and therefore single silver atoms may be induced in the silver halide crystals by transient exposure to an electronic flash. The duration of the flash and its intensity (varied by changing the distance between film and electronic flash-gun) is critical and should be determined empirically. Set the shortest duration on the flash-gun and position the gun at varying distances (increasing in steps of 1 m) from film test strips. Develop the film (and a piece of unflashed original) and use the conditions which just give barely visible fogging.

Exposure of the gel with intensifying screens and preflashed film at –70°C gives maximal stabil- ity of both the nascent image and the sensitized preflashed areas.

• It is convenient to use a mixture of purified radioactive proteins as molecular weight markers

in these gels. Suitable mixtures for different fractionation ranges may either be bought com-

mercially as a 14 C-methylated protein mixture or prepared from an ‘in-house’ mix of purified

4.10AUTORADIOGRAPHY AND FLUOROGRAPHY

• Much has been written about the artefacts associated with autoradiography. The two most commonly seen are: (a) A homogeneous fogging of the film, often in areas away from contact with the gel. This

is due to chemical fogging and probably means that the acetic acid failed to volatilize during drying. Dry the gel with a hot air drier and re-expose to X-ray film.

(b) Lightning strikes. This is caused by an electrostatic discharge from either your fingers or dampness causing the surfaces of the gel, screen or film to adhere. When you peel the sur- faces apart an electrostatic charge is generated. Earth your fingers by touching the bench and make sure the interior of the cassette is warm and dry aespecially if it has been exposed at –70°C abefore you open it.

• If your cassettes are used communally, it is a wise precaution to line them with plastic benchcote or candy paper. If one becomes contaminated then the liner rather than the cassette can be replaced. Similarly, it is good practice to clean the intensifying screens regularly with an anti- static screen cleaner.

• As noted in Fig. 3.15, this technique is usually used qualitatively; however, it is relatively simple to quantify the radioactivity contained in the gel, although there is considerable loss of sensitivity. If you intend to measure the radioactivity, it is helpful to stain the gel, prior to drying the gel down. This will help you to locate the individual tracks. Remember that quantification can be carried out after the gel has been exposed to X-ray film, provided that sufficient radioactivity remains. (a) Lay the stained, dried gel on a glass plate and cut it up into its individual lanes using a scalpel

blade and straight edge. (b) Cut strips of 1-mm tracing graph paper approximately the same size as the gel tracks. (c) Use spray adhesive to attach a strip of graph paper to each gel track, at right angles to the

polypeptide bands. (d) Cut each track into 1-mm strips using a sharp pair of scissors, place each strip in a g spec- trometer and plot a graph of the amount of radioactivity. (e) Count in a g spectrometer and plot a graph of the amount of radioactivity against the dis- tance travelled down the gel. (f) Compare the result with that obtained from the X-ray film.

4.10.2 Fluorography

Weak α and β emitters would be detected very inefficiently by direct autoradiography because of the relatively short distances travelled by these radiations. Instead a scintillant is incorporated directly into the gel and the energy from the decay of the radioisotope is transferred to the X-ray film as photons of light.

In the original method for gel impregnation, the scintillant (usually 2,5-diphenyloxazole a PPO) was dissolved in dimethyl sulphoxide (DMSO), thus causing considerable shrinkage and distortion of the gel. Aqueous preparations are now commercially available (e.g. Amplify, APBiotech; see Appendix C. In both cases they are available as liquid or spray). The gel is fixed in an aqueous solution of methanol–acetic acid as above and then soaked in the scintillant solution.

C H A P T E R 4: Antibodies as probes

After drying (without glycerol), the gel is loaded into a metal cassette with X-ray film and exposed at –70°C.

4.10.3 Ultrasmall gold probes

The advantage of labels such as enzymes is that they amplify the reaction and lead to an easily visible product. In so doing they can lead to some diffuseness with loss of structural detail. Gold provides a useful label where this fine structure is critical. Colloidal gold particles can be coupled to proteins and so used as the label coupled to antibodies or detection molecules, such as protein

A. Best resolution is achieved using small particles (> 1 nm), and then enhancing the detection by deposition of silver on top of the gold. Gold-coupled reagents are available commercially together with silver enhancement systems. These may be visualized with a light microscope, an electron microscope and in protein blotting.