Rapid scanning of 32P-labeled acrylamide gels

ANALYTICAL

BIOCHEMISTRY

53,

272-277 (1973)

SHORTCOMMUNICATIONS

Rapid

Scanning

of

32P-Labeled

Acrylamide

Gels

A simpleand rapid procedurefor scanningand quantitative evaluation of acrylamidegelscontaining“P-labeled material is described.It doesnot require any processingof the gel.

During an investigation of autodigestion of ribosomal RNA in intact E. coli ribosomes we have used the technique of acrylamide gel electrophoresis to monitor t.he extent of fragmentation of 32P-labeled ribosomal RNA. Since slicing gels (l-3) and counting individual slices was a limiting factor in this study the simple autoradiographic procedure described here was devised to permit rapid scanning of large numbers of radioactive gels. This procedure is analogous to but simpler than the autoradiography of dried gels (67). However, it is applicable only to 32P-labeled material. Results obtained by this method and by slicing the gels and counting the slices are equivalent qualitatively and quantitatively. It is hoped that, this rapid and simple technique will be useful to other investigators confronted with similar problems. MATERIALS AND METHODS

The preparation and incubation of 32P-labeled E. coli ribosome subunits under conditions which lead to various degrees of RNA fragmentation will be described elsewhere (8). Incubation samples are heated at 90°C for 2 min in the presence of 11% w/v sodium dodecyl sulphate to dissociate ribosomal protein and RNA and to reveal “hidden” breaks in the ribosomal RNA, and are submitted without further treatment to electrophoresis in 3% acrylamide gels (9). Gel columns, prepared in 6 mm internal glass tubes are 8 cm long. The autoradiography apparatus consists of a rectangular perspex tray and cover (Fig. la,lb) and a sheet. of polyethylene (Fig. lc). The tray is made by machining a 3 X 20 mm wide rectangular rebate along one long edge of a 40 x 15 X 0.8-cm perspex slab, and a series of 3 X 9.5-mm rectangular slots across the same side of the slab. The rebate is then refilled by glueing a 3 X 20-mm perspex fillet into it. In this 272

Copyrigtit @ 1973by AcademicPress,Inc. AU righta of reproduction in any form reserved.

SHORT

COMMUNICATIONS

273

FIG. 1. Construction of autoradiography apparatus (dimensions are in millimeters). (a) Perspex tray with the slots; (b) perspex cover; (c) polyethylene sheet.

way a series of flat-ended rectangular slots are obtained. A second 40 x 15 X O&cm perspex slab and a thin sheet of any water-resistant disposable material (we use 0.2-mm polyethylene sheet) of similar dimensions are used to cover the tray. As shown, the apparatus permits simultaneous autoradiography of 13 gels. Its length was chosen to correspond to the longer dimension of standard x-ray films. With the dimensions given, the slots in the tray have the same cross-section as acrylamide gels prepared in 6-mm internal diameter glass tubes. Gels are placed in the slots of the tray with the top in contact with the flat end of the slot. Plasticine plugs are placed in the slots at the other end of each gel to ensure that gels do not elongate when compressed in the assembled apparatus. The polyethylene sheet is placed over the loaded tray and is covered in turn (in a dark room) by a 40 X 15-cm piece of Kodak RPSX-omat film, and the perspex cover slab and the whole assembly is compressed by placing bulldog clips round its edges. The gel columns thereupon become rectangular in section and fill the slots in the tray completely. After a suitable exposure time the film is removed and developed. Quantitative estimates of the relative intensities of radioactive bands in the gels are obtained by cutting out the rectangles of film corresponding to each gel and scanning them in a microdensitometer (Joyce Loebl Chromoscan). For scanning by conventional methods gels are frozen and sliced into 2-mm slices which are counted in a gas flow

274

SHORT

COMMUNICATIONS

or scintillation counter. The ratios between disintegrations per minute present in a single gel slice and the counts per minute measured in the same slice in a gas flow counter or by Cerenkov radiation in a scintillation counter were 0.25 and 0.40, respectively. RESULTS

AND

DISCUSSION

When the time of exposure of the x-ray film is correct, a good correlation is found between the microdensitometer scan of the film and the profile obtained by slicing and counting the gel. Figure 2 shows a calibration curve obtained by autoradiography of 1 cm long gel columns containing 2-20 X lo5 dpm of 32P orthophosphate uniformly distributed throughout their volume, and measurement of the optical densities of the

20

0

0

0.1

0.2

0.3

0.4

05

Bond

opt~col

0.6

0.7

0.8

a9

densfly

FIG. 2. Darkening of x-ray film produced by 05hr exposure to 10 x 6-mm diameter gel segments containing various levels of uniformly dispersed inorganic “‘P. Each gel segment was prepared in triplicate and several radioautographs were made from each sample. The optical densities found fell within ranges indicated by the horizontal bars. The standard deviation was estimated to be 0.18 and 0.23 for two levels of radioactivity.

SHORT

COMMUNICATIONS

275

corresponding regions of an x-ray film exposed for 30 min. It is possible to deduce from this curve an approximate exposure time for a gel column upon which a known amount of 3”P-labeled material has been electrophoresed. Allowance must be made for the expected dispersion of this material in the gel column, i.e., for the number of different molecular species in the original material. For example consider the case of the gel column whose 32P RNA distribution profiles obtained by autoradiography, and by slicing and counting individual slices, are compared in Fig. 3. The RNA sample (5 X lo6 dpm of ,‘P) subjected to electrophoresis in this gel was prepared from “2P-labeled E. coli 50 S ribosome subunits which had been subjected to autodigestion for 24 hr at 40°C. It was known to contain material ranging in size from 23 S to about 4 S. Assuming a completely uniform distribution of the radioactive material after electrophoresis, this gel column (8 cm long) will contain 0.6 X lo6 dpm per cm. Figure 2 shows that for an exposure time of 30 min, blackening of the exposed film is proportional to t,he 32P content per cm of gel up to a limit of about 1.8 X lo6 dpm. Hence the exposure time for a gel column containing 0.6 X lo6 dpm per cm should not

FIG. 3. Comparison of distribution profiles of 32P obtained by autoradiography, and by slicing an’d csunting individual slices. Distance of migration is measured from the. top.ef tie gel. (-) Absorbance of the exposed x-ray film (white light) (- - - - -) “P dpm per Z-mm gel slices.

;

276

SHORT COMMUNICATIONS

exceed 90 min. An exposure time of 45 min was therefore chosen. The densitometer tracing of the developed film (Fig. 3) shows that a fairly constant ratio exists between the amount of radioactivity in a gel slice and the darkening produced by the corresponding region of the gel column for all except the slowest moving component (23 S) for which the scan shows a plateau (first peak Fig. 3). Two millimeter gel slices in this peak contained 0.460.45 X lo6 dpm, i.e., the peak region contained 2-2.3 X lo6 dpm per cm and at this level of radioactivity as shown in Fig. 2 darkening of the x-ray film is no longer proportional to the amount of radioactivity for a shorter exposure time of 30 min. It must be pointed out that this technique is limited in so far as it is not possible to estimate quantitatively on the same film the relat.ive amounts of radioactivity in two gel bands if these amounts differ too widely, i.e., by a factor greater than about lo-fold. It is however POSsible to use two different times of exposure and an additional band of intermediate intensity (existent in the gel or added graphically to the film strips) as a standard for comparison. This technique can be considered as a complement to a previously described (4-7) in which gels are sliced longitudinally and dried before radiography. It permits rapid localization and quantitative estimation of s2P-labeled components in a gel without cutting and drying a longitudinal slice, both delicate operations in the case of gels of low acrylamide concentration. ACKNOWLEDGMENTS This work was supported de la Recherche Scientifique It la Recherche Scientifique pour la Recherche M&l&de The authors wish to thank

by grants to D. H. Hayes from the Centre National (Equipe de Recherche no lOl), la Dkl6gation G&u%-ale et Technique (convention no 70 02 251), la Fondation Franpaise, and le Commiasariat $ 1’Energie Atomique. Mr. C. Le Gal for building apparatus.

REFERENCES 1. CHRAMEACII,

A. (1960) Anal. Biochem. 15, 544. C. A., EDGELL, M. H., AND SINSHEIMER,

R. L. (1997) J. Mol. Bol. 23, 553. 3. IAN~LO, J. J. (1970) And. Biochem. 36, 6. 4. FAIRBANKS, G., JR., LEVINTHAL., C., AND REEDER, H. R. (1965) B&him. Biophys,

2. HUTCHINSON,

Res.

Commun.

20,

393.

5. REID, M. S., AND BIELESKI, R. L. (1998) Anal. Biochem. 22, 374, 6. LIM, R., HUANQ, J. J., AND DAVIS, C. A. (1969) Anal. Biochem. 29, 48. 7. DANIELS, M. J., AND WILD, D. G. (1970) And. Biochem. 35, 546.

S. BARRITAULT D. H. HAYES D.

SHORT

COMMUNICATIONS

Institut de B&lo&e Physico-Chimique 13, Rue Pierre et Marie Curie Paris &me-France Received April 5, 1972; accepted December

11. 1912

277