[7] AUTORADIOGRAMS: 35S AND 32p 55

[7] A u t o r a d i o g r a m s : a s s a n d 32p
By WILLIAM M. BONNER

Autoradiography in the strict sense refers to the exposure of film by

radioactive particles, whereas fluorography refers to the exposure of film
by light generated from the interaction of radioactive particles with added
compounds called fluors. Whether fluors are mixed with or are external to
the sample depends on the energy and range of the emissions.
Data and comments concerning the relative and absolute sensitivities
of the different methods to be described are presented in Table I. The
absolute sensitivities are based on the minimum film darkening (0.02 A540)
detectable in 24 h r ) -a Note that it may take 10-100 times these amounts
to get a good exposure overnight. The procedures given below for 35S also
apply to 14C and 32p. For 3H, see Bonner and Laskey.l-3

3ss Autoradiography
Because of the short range of 35S fl rays (0.25 mm in water or plastic),
careful consideration of several factors is necessary for efficient autora-
diography. Gels should be prepared as thin as possible and should be fixed
or soaked to remove nonvolatile solutes such as urea so that the gel can be
dried as thin as possible. Soaking DNA sequencing gels (0.35 mm thick
acrylamide) in 5% methanol-5% acetic acid for 15 min before drying
increases the autoradiographic efficiency 3-fold. 4 After removing any
plastic cover used during drying (see below), the gel is pressed firmly
against a piece of Kodak X-Omat AR film and exposed at room tempera-
ture. Wet gels, covered with thin plastic wrap, can also be exposed this
way but with a very low efficiency. Blots are dried in air or vacuum and
the surface containing the radioactivity is placed against to the film.

3ss Fluorography
Since fluors must be impregnated into the gel matrix for 35S fluorogra-
phy, the main consideration besides cost is whether the procedures will
lead to a significantloss of resolution or material due to diffusion.Impreg-

l W. M. Bonner and R. A. Laskcy, Eur. J. Biochem. 46, 83 0974).

2 R. A. Laskey, this series, Vol. 65, p. 363.
3 W. M. Bonncr, this series, Vol. 104, p. 460.
4 D. L. Ornstein and M. A. Kashdan, BioTechniques 3, 476 0986).

METHODS IN ENZYMOLOGY,VOL. 152

56 ISOLATING AND CHARACTERIZING NUCLEIC ACIDS [7]

TABLE I
SENSITIVITIES OF DIFFERENT METHODS OF 358 AND 32p FILM DETECTIONa

Technique Temperature Sensitivity

and sample (°C) (dpm/mm 2) Comments

35S Autoradiography
Gels +20 20-60 Depends on gel thickness, solute
concentration. Wet gels give
much lower sensitivity
Blots +20 20 Radioactivity on the surface
35S Fluorography
Gels/blots -70 4 Small molecules could be lost
during impregnation
32PAutoradiography
Gels/blots +20 5-10 Wet gels give less sensitivity and
resolution. Two sheets of lead
enclosing film-sample double
sensitivity
32pFluorography
Gels/blots -70 1 Screen-film-sample
Gels/blots -70 0.5 Screen-flashed film-sample-lead
(or screen)
Gels/blots +20 1 Screen-flashed film-sample-lead
(or screen). Wet gels give less
sensitivity and resolution

a These sensitivities are based on the minimum darkening (0.02 A540)detectable in 24 hr.
A good exposure overnight may require 10-100 times these amounts. Film is Kodak
X-Omat AR film developed by machine. Screen is Cronex Lightning Plus. Lead is a
sheet of lead foil. Dry gels were used to compare sensitivities.

nation procedures depend on the type of gel as well as the type of fluorog-
raphy solution.
Acrylamide gels may be stained, fixed with 10% acetic acid/0-30%
alcohol, or prepared directly for fluorography. They may be treated with
the original 2,5-diphenyloxazole-dimethyl sulfoxide (PPO-DMSO) solu-
tion, 1-3 commercial water-insoluble products, or the more recently devel-
oped water-soluble products which process the gel in 15-30 min in one
step. Some examples of the latter group are Amplify (Amersham),
E N L I G H T N I N G (New England Nuclear), Fluoro-Hance (Research
Products International Corp.), and Autofluor (National Diagnostics). All
these methods yield roughly comparable results with fixed gels, but ease
of use and cost vary widely. Gels are dried as described below.
In one protocol, DNA sequencing gels are fixed in 5% methanol-5%
acetic acid for 15 min, soaked for 30 min in EN3HANCE autoradiography
[7] AUTORADIOGRAMS; 35S AND 32p 57

enhancer (New England Nuclear), and washed in cold water for 15 min to
precipitate the fluor in situ before drying. Enhancement is 4-fold for large
oligonucleotides, but minimal for small ones, perhaps due to losses during
soaking.
Agarose gels are soaked first in absolute ethanol for at least 30 min to
remove water and solutes, then in a 3% (w/v) solution of PPO in ethanol
for 3 hr. 5 The gels are then soaked 1 hr in water to precipitate the 2,5-di-
phenyloxazole in the gel before drying. The commercial water-soluble
reagents listed above can also be used for agarose gels.
Blots can be treated by several methods. (1) The dried filters are
dipped in a 20% (w/v) solution of PPO in toluene and redried. 6 (2) The
dried filters are dipped in slightly warmed (40°) 0.4% PPO in 2-methyl-
naphthalene, drained, and cooled. The 2-methylnaphthalene solidifies at
340.7 (3) The dried filters are sprayed with the commercial spray reagent
EN3HANCE and redried.
After preparation and drying (see below), the fluor-impregnated sam-
ple is exposed to the flashed face of a piece of Kodak X-Omat AR film and
exposed at - 7 0 °.

a2p Autoradiography and Fluorography

The fl ray of 32p has a range of about 6 mm in water or plastic, enabling
external fluors in the form of intensifying screens to be used. The major
consideration in choosing a 32p detection procedure is whether the sample
can be frozen.
Freezable samples include dried acrylamide or agarose gels, and wet
or dried blotting membranes. Acrylamide or agarose gels can be dried
immediately after electrophoresis (see below). The sample, which can be
wrapped in a plastic folder, is placed next to a piece of flashed Kodak
X-Omat AR film, with a CaWO4 intensifying screen (Cronex Lightning
Plus, DuPont) on the other side of the film (flashed face of film toward
screen). 8,9 A second CaWO4 screen or a sheet of lead foil about 0.15 mm
thick can be placed behind the sample for approximately 50% increase in
speed. This second screen or lead sheet acts by increasing the backscatter
of 32p/~ rays from the sample rather than by any fluorographic process.
Expose at - 7 0 °.
Thus the most sensitive arrangement is screen-flashed film-sample-

5R. A. Laskey, A. D. Mills, and N. R. Morris, Cell (Cambridge, Mass.) 10, 237 0977).
6 E. M. Southern, J. Mol. Biol. 98, 503 0975).
7 W. M. Bonncr and J. D. Stcdman, Anal. Biochem. 89, 247 (1978).
8 R. A. Laskey and A. D. Mills, FEBS Lett. 82, 314 0977).
9 R. Swanstrom and P. R. Shank, Anal. Biochem. 86, 184 0978).
58 ISOLATING AND CHARACTERIZING NUCLEIC ACIDS [7]

screen (or lead) (Table I). The flashed film and second screen or sheet of
lead behind the sample each result in a 50% increase in sensitivity for the
fainter bands, even during hour long or overnight exposures. Increases in
sensitivity may be considerably larger on weeklong exposures, but flashed
film may become too fogged on long exposures with some screens. 2 An-
other arrangement sometimes seen, screen-film-screen-sample, has
lower resolution and about half the sensitivity as the arrangement de-
scribed above.
Nonfreezable samples such as wet gels are exposed as described for
dried gels except at temperatures above 40.8 Flashed film and screens are
more important for adequate sensitivity above 4 °. However, resolution
and sensitivity will be less because the sample is thicker. If the wet gel
contains sufficient radioactivity to be detected by autoradiography alone
(no screens or flashed film), then a convenient procedure is to seal the gel
in a plastic bag and lay it on an envelope of "ready pack" film (individu-
ally wrapped and sealed). No darkroom is needed until the film is de-
veloped.

Drying Gels
To minimize tearing and creasing of the gel during drying, lay it on a
thin flexible plastic sheet such as Mylar, let it relax, then lay the paper
backing (Whatman 1 or equivalent) on top of the gel, and roll out any air
bubbles. Alternatively, 32p-labeled DNA sequencing gels can be left on
one glass plate and a sheet of backing paper pressed onto the gel. The
backing paper is peeled off bringing the gel with it. The opposite face of
the gel is covered with a thin plastic wrap. Then in either case, several
sheets of heavier paper (Whatman 3 MM or equivalent) are added next to
the backing paper as a support pad and the sandwich is put into the gel
dryer (paper pad closest to vacuum source). Acrylamide gels are dried
under vacuum at 60-80 ° (about 1 hr for a 0.8-mm-thick gel). Agarose gels
are dried under vacuum with gentle or no heat to prevent melting. Gels
containing fluors should not be dried or heated for excessively long times
because these compounds are somewhat volatile.
The main problem usually encountered during drying is fracturing of
acrylamide gels. This can be avoided if the gels are prepared so that the
product of the final concentrations of acrylamide and bisacrylamide (ex-
pressed as percentages) equals 1.31°; this ensures adequate gel elasticity.
In addition, the vacuum should not be released before the gel is com-

10 D. P. Blattner, F. Garner, K. Van Slyke, and A. Bradley, J. Chromatogr. 64, 147 (1972).
[7] AUTORADIOGRAMS: 35S AND 32p 59

pletely dried. Acrylamide gels may be dried without a gel dryer if they
are bonded during polymerization to one of the glass plates 11or to a com-
mercial product such as Gel Bond film (FMC Corporation, Rockland,
ME).
For best results, commercial gel dryers should be connected to a
corrosion-resistant mechanical vacuum pump with an acid-base vapor
trap and a cold trap rather than to a house or aspirator vacuum. Commer-
cial lyophilizers will work but may be damaged in such service.

Cassettes, Screens, Films, and Indexing Marks

Intimate juxtaposition of the sample, film, and screens is important for
the best sensitivity and resolution. For 35S autoradiography in particular,
the sample should be dried as thin as possible, any plastic covering mate-
rial used in drying the gel should be removed, and the dried gel must be in
direct uniform contact with the film emulsion. For 32p, the wet or dry gels
or filters can be wrapped in plastic, taking care to remove any trapped air
pockets that might interfere with good uniform contact between the film,
sample, and screens. Sometimes, old screens may become wavy, leaving
areas of fuzzy bands due to poor contact.
For indexing, a 14C-labeled ink (1 /zCi/ml) can be used on top of the
plastic (next to the film surface) or a 32p-labeled ink can be prepared for
use inside the plastic (use Table I as a guide to the amounts of radioactiv-
ity to use). A convenient alternative to radioactive ink is a nonradioactive
phosphorescent marker (UltEmit, New England Nuclear). The markings,
when activated by ambient light, glow for approximately 2 hr, thus trans-
ferring their images to the film.
A commonly used and widely available film-screen combination is
Kodak X-Omat AR film (blue-sensitive screen type) and Cronex Light-
ning Plus screen (CaWO4, blue emitting). Only one screen need be used
per cassette, as a thin sheet of lead (about 0.15 mm or 0.006 in. thick) can
substitute for the screen behind the sample.
Other film-screen pairs can be used and may be just as sensitive as the
above, but for maximum efficiency the film sensitivity must match the
screen emission spectrum. One example of a green-emitting screen is
Lanex (gadolinium lanthanium oxysulfide; Eastman Kodak Co.) which is
paired with a green-sensitive film, Kodak Ortho H. When choosing a
screen, check with the manufacturer for the most sensitive compatible
film. References 8 and 9 compare several screen-film combinations.

11 H. Garoff and W. Ansorge, Anal. Biochem. 115, 450 (1981).

60 ISOLATING AND CHARACTERIZING NUCLEIC ACIDS [7]

Film Development
Film can be stored at room temperature or at 4°, but it should be
protected from radiation. Kodak X-Omat AR film can be obtained in
individually wrapped "ready packs," which have the advantage that
other sheets of film are not inadvertently exposed while removing one
sheet. Most X-ray departments have automatic processing equipment for
X-Omat films, and it may be possible to make an arrangement with one
nearby for developing films. The film can be removed immediately from a
cassette at - 7 0 °, but cold cassettes will quickly collect moisture from the
air so it may be necessary to let them warm up before inserting a second
film.
Manual processing can be carried out with four trays in a dark room
with a red safelight for blue-sensitive films like Kodak X-Omat AR, or a
dark red one for green-sensitive films. For Kodak X-Omat film at 20°, the
procedure is to develop for 5 min, rinse for 30 sec, fix for 2-4 min, wash
for 5 rain in clear running water, and dry in a dust-free area. The chemi-
cals are inexpensive and easily obtained. If a small area can be set aside
for a darkroom, then a tank with three chambers can be set up with a
water line temperature mixing valve and rust filter for a permanent man-
ual developing system. A film dryer can be set up either in or just outside
the darkroom.

Preflashed Film
The sensitivity of film to light is nonlinear. Flashing the film sensitizes
it to low intensities of light, making its response more linear.12
A film flasher can be made from an inexpensive battery-operated cam-
era flash unit. The unit needs to have a flash duration less than 1 msec, a
button so that it can be triggered without a camera, and a flash face that
can easily be covered with filters. The light output is greatly decreased by
covering the flash face with an orange Kodak Wratten 21 or 22 filter and
layers of diffusing neutral density filters such as heavy white paper or
partially exposed film, and by blocking off some of the flash face with
opaque black paper.
Flashing is done in total darkness. The film is laid on the floor on a
yellow backing sheet and the flash unit held approximately eye level. One
flash should give a fogging density in the developed film of approximately
0.15 A540 (measured in a spectrophotometer by cutting pieces of film to fit
a cuvette holder). Fine adjustments can then be made by changing the

t2 R. A. Laskey and A. D. Mills, Eur. J. Biochem. 56, 335 (1975).

[8] GEL ELECTROPHORESIS 61

height of the flash unit or by adding or deleting filters. The flashed face of
the film should face the fluor, in the sample for 35S, in the screen for 32p.

Quantitation of Autoradiographs
Film response to autoradiography (no fluors or screens) is linear up to
absorbances of about 1 unit. With 35S autoradiography, it is important that
the gel matrix be uniform, because different regions of a gradient gel could
quench asS to different extents. Even though the nonlinear film response
in fluorography can be corrected by preflashing the film, linearity should
be checked with standards prepared with the same isotope.

[8] E l e c t r o p h o r e s i s in A g a r o s e a n d A c r y l a m i d e G e l s
By RICHARD C. OGDEN and DEBORAH A. ADAMS

The scope of this chapter is to present a range of methods by which

DNA and RNA molecules can be fractionated and analyzed by means of
gel electrophoresis. This chapter will emphasize those techniques which
can be simply and routinely applied in the course of molecular cloning and
analysis and, wherever appropriate, reference will be made to more ex-
haustive practical or theoretical considerations of the techniques.
Gel electrophoresis through agarose or polyacrylamide is a very pow-
erful method for rapidly resolving mixtures of nucleic acid molecules
which has found wide application in recombinant DNA research. The
resolution afforded far exceeds that generally obtained by other sizing
techniques. The fractionated nucleic acids can be directly "viewed" in
situ in the gel and can be readily recovered by a variety of methods
tailored to subsequent steps in an experimental protocol. Because it is
such an indispensible technique, a great deal of effort has gone into im-
proving its efficacy for particular applications, with the result that many
of the original methods have been simplified, scaled down, and improved.
This review will cover the various choices to be made when confronted
with experiments requiring gel electrophoresis in the order they would
typically arise, starting with the type of gel system, choice of running
buffer, and equipment, and continuing through gel running, visualization
of the separated molecules, extraction of the material from the gel, and
workup of preparative samples. Certain specific applications of electro-
phoresis, for example gels for sequencing purposes (this volume [56, 57])

Copyright © 1987 by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 152 All rights of reproduction in any form reserved.