A Film Detection Method for Tritium-Labelled Proteins

Transcription

1 Eur. J. Biochem. 46, (1974) A Film Detection Method for Tritium-Labelled Proteins and Nucleic Acids in Polyacrylamide Gels William M. BONNER and Ronald A. LASKEY Medical Research Council Laboratory of Molecular Biology, University Postgraduate Medical School, Cambridge (Received March 5/April 12, 1974) A simple method is described for detecting 3H in polyacrylamide gels by scintillation autography (fluorography) using X-ray film. The gel is dehydrated in dimethyl sulphoxide, soaked in a solution of 2,5-diphenyloxazole (PPO) in dimethylsulphoxide, dried and exposed to RP Royal X-Omat film at -70 C. Optimal conditions for each step are described.,&particles from 3H interact with the 2,5-diphenyloxazole emitting light which causes local blackening of an X-ray film. The image produced resembles that obtained by conventional autoradiography of isotopes with higher emission energies such as 14C dis. 3H/min in a band in a gel can be detected in a 24-h exposure. Similarly 500 dis./min can be detected in one week. When applied to the detection of s5s and l4c in polyacrylamide gels, this method is ten times more sensitive than conventional autoradiography. 130 dis. 3% or 14C/min in a band in a gel can be detected in 24 h. Autoradiography has been widely used to dettec labelled compounds after electrophoresis in polyacrylamide gels. It is simple, rapid and does not involve destruction of the sample, but its use has been restricted to such isotopes as 14C, 35S, 32P, l25i and In spite of its lower emission energy, 3H remains the most satisfactory isotope for a wide range of experiments because a wide range of 3H-labelled precursors is available at high specific radioactivity. Hitherto efficient detection of 3H in gels has required scintillation counting of dissolved gel slices, a timeconsuming procedure which affords limited resolution and destroys the sample. Wilson [l] showed that the detection efficiency for 3H on paper chromatograms can be increased by impregnating the chromatogram with a scintillator before applying film. Thus the film is not exposed by /%particles themselves, but by light generated by interaction of the b-particles with 2,5-diphenyloxazole (PPO). Randerath [2] has studied the factors which contribute to the efficiency of this process (fluorography or scintillation autography). He has shown that treatment of thin-layer chromatograms with a solution of PPO in ether permits efficient detection of 3H-labelled nucleotides by medical X-ray film. Until Abbreviations. EMC, encephalomyocarditis; MeZSO, dimethyl sulphoxide; PPO, 2,5-diphenyloxazole. now this process has only been successfully applied to molecules situated on or near surfaces and not to molecules inside the lattices of polyacrylamide gels. In this paper we describe a fluorographic method for detection of 3H-labelled molecules inside polyacrylamide gels. MATERIALS AND METHODS Muter iuls Seakem Agarose was obtained from Micro-Bio Laboratories Ltd. PPO (Scintillation chemical grade) and N,N,N,N -tetramethylethylenediamine were purchased from Koch-Light Laboratories Ltd (Colnbrook, Bucks, England). All other reagents, including dimethyl sulphoxide (MezSO) were analytical reagent grade (AnalaR) and purchased from BDH Chemicals Ltd (Poole, Dorset, England). [35S]Methionine (156 Ci/ mmol), [U-14C]valine (256 mci/mmol), [5-3H]proline (10 Ci/mmol), [2,3-3H]valine (39 Ci/mmol) and [5-3Hluridine (25 Ci/mmol) were purchased from the Radiochemical Centre (Amersham, Bucks, England). Medical X-ray films were purchased from Kodak Ltd (London). Omnispray aerosol and Aquasol were purchased from New England Nuclear.

2 84 Fluorographic Detection of 3H, 14C and 35S in Acrylamide Gels Labelling of Proteins and Fractionation in 15% Polyacrylamide Gels 15 % polyacrylamide slab gels containing 0.09 % bisacrylamide and 0.1 % sodium dodecylsulphate were prepared and electrophoresed using Laemmli s discontinuous buffer system [3] as described previously [4]. When stained gels were required, a solution of 0. I % Coomassie blue in 30 % methanol - 10 % acetic acid was used. Gels were destained in 30% methanol -10% acetic acid. To test the procedure for detecting 3H, 35S or 14Clabelled proteins in gels, proteins labelled with [3H]- proline, [3H]valine, [14C]valine or [35S]methionine were prepared by translating encephalomyocarditis (EMC) viral RNA in oocytes of Xenopus laevis as described previously [4], a procedure which yields a series of EMC proteins of known molecular weights in addition to some oocyte proteins. Labelling of Nucleic Acids and Fractionation in 2.2 % Polyacrylamide Gels Containing 0.5 % Agarose Slab gels containing 2.2 % acrylamide, 0.11 % bisacrylamide and 0.5 % agarose were prepared and electrophoresed by the procedure of Peacock and Dingman [5], substituting % N,N,N,N -tetramethylethylenediamine for dimethylaminoproprionitrile. 3Hlabelled ribosomal RNA used to test the detection procedure was extracted by Miller and Knowland s pronase method [6] from 3T6 mouse fibroblasts which had been labelled with [5-3H]uridine. Drying of Gels Polyacrylamide gels were dried in approximately one hour by evacuating a polythene bag containing the gel supported on Whatman 3 MM paper on a porous polythene pad. The evacuated bag was placed over a boiling water bath so that the surface covering the gel was continuously exposed to steam. Detection of Radioactivity Details of the fluorographic procedure are presented in Results. The amounts of radioactivity applied to gels were determined by counting aliquots either in solution in Aquasol (New England Nuclear) or on filters in 4 g PPO/l toluene. The amounts of radioactivity in slices of dried gels were determined by dissolving in 19 parts 30 % HZOZ and 1 part 0.88 ammonia at 37 C followed by counting in Aquasol. Counting efficiencies were determined by the channels ratio method and were 20 (&3)% for 3H and 80-85% for 35s and 14C. RESULTS AND DISCUSSION In the method described here the gel is impregnated with a scintillator, PPO, so that the sensitive method of fluorography can be used to detect 3H. Because PPO is insoluble in water, it was necessary to find a solvent which dissolves PPO and which can replace water in the gel without causing distortion. Dimethyl Fig. 1. A 6-h fluorographic exposure oja 15 %polyacrylamide gel containing 3H-labelled proteins. Proteins labelled with [5-3H]proline were obtained from oocytes injected with EMC viral RNA as described in Methods counts 3H/min of labelled proteins were electrophoresed in a 9-mm slot in a slab gel containing 15% acrylamide and 0.1 % sodium dodecylsulphate and the gel was processed for fluorography by the optimal procedure described in the text. RP Royal lo% Molecular weight X-Omat film was exposed to the gel at - 70 C for 6 h. This figure shows a photograph of the developed film and a trace obtained by scanning the gel with a Joyce-Loebl microdensitometer. The peaks of and molecular weight contained counts and 6000 counts 3H/min respectively, as determined by scintillation counting of dissolved slices of the gel

3 W. M. Bonner and R. A. Laskey 85 Step 2. Immerse the gel in 4 volumes of 20% (w/w) PPO in MezSO (22.2%, w/v) for 3 h. Step 3. Immerse the gel in 20 volumes of water for 1 h. Step 4. Dry the gel under vacuum (1 h). Step 5. Place RP Royal X-Omat or equivalent Medical X-ray film in contact with the dried gel and expose at -70 C. (Caution : because of the known skin-penetrating properties of MezSO we suggest that rubber gloves should be worn when solutions of PPO in MezSO are handled). This procedure may be used on unfixed, fixed or stained gels. Prolonged exposure to MezSO removes Coomassie blue from protein bands with the result that faintly stained bands may not be visible at the end of the procedure. Gels which have already been dried must be swollen in water or fixative before proceeding to MezSO. Gels may be frozen for storage at any stage of the procedure. 0 Migration Fig. 2. Fluorographic detection of [3H]RNA in a 2.2% acrylamide gel containing 0.5 % agarose. RNA labelled with [5-3H]uridine was obtained from 3T6 mouse fibroblasts and electrophoresed in a 2.2% acrylamide slab gel containing 0.5% agarose as described in Methods counts [3H]RNA/min were electrophoresed in a 12-mm slot and the gel was processed for fluorography as described in the text. RP Royal X-Omat film was exposed to the gel at - 70 C. A photograph of an 18-h exposure and a microdensitometer trace of a 5.5-h exposure are shown. 4-S and 5-S RNA have migrated off the end of the gel sulphoxide (MezSO) was found to be satisfactory in both respects. Examples of fluorographs obtained from 3Hlabelled macromolecules in pclyacrylamide gels are shown in Fig.1 and 2. The method is seen to apply equally to 3H-labelled proteins electrophoresed in high acrylamide concentrations and to -labelled RNA electrophoresed in low acrylamide concentrations. Optimal Procedure The optimal procedure for this method is as follows : Step I. Directly after electrophoresis or after staining, soak the gel in about 20 times its volume of MezSO for 30min, followed by a second 30-min immersion in fresh MezSO. 0 Discussion of the Procedure Step I. PPO is very soluble in MezSO but insoluble in water or water -MezSO mixtures. Therefore water must be removed from the gel before introducing the PPO. Two incubations in excess MezSO were found to be sufficient and the two MezSO baths can be reused in the same sequence. Step 2. For 3H detection in 15 % gels the greatest sensitivity was obtained when gels were incubated in an excess of 16 % (w/w) PPO in MezSO for 3 h (Fig. 3). Similar optimum conditions were found for 9% gels and for 2.2 % gels containing 0.5 % agarose, though the optimum peak was slightly broader in the last case, extending from % (w/w). The similarity between the optimum conditions for 15 % and 2.2 % gels suggests that 16 % (w/w) may be the optimum PPO concentration for most of the gel concentrations in common use. Because incubation of the gel in a large excess of 16 % PPO is inconvenient, an alternative procedure has been adopted, i.e. the gel is soaked in 4 volumes of 20% (w/w) PPO in MezSO (22.2%, w/v) which gives a 16% w/w (17.8% w/v) solution after equilibration with the gel. A concentration between 14% and 19% (w/w) yields an efficiency of at least 80% maximum. If the procedure we recommend is used the solution should be used only once since further use would lower the PPO content below the optimum. For a slab gel 14cmx15cmx1.5mm, about 6g PPO enters the gel. PPO from the excess solution can easily be recovered for re-use by precipitation with water. Steps 3 and 4. After step 2 the PPO-impregnated, but undried, gel can be used directly for fluorography.

4 86 Fluorographic Detection of 3H, 14C and 35S in Acrylamide Gels.- x c a, PPO concentration (%,w/w) Fig. 3. Optimum conditions of PPO impregnation for detection of 3H in polyacrylarnide gels. Proteins labelled with [3H]proline were extracted from 3T6 mouse fibroblasts and mixed with acrylamide before polymerisation of a 15 % slab gel, ensuring an even distribution of 3H through the gel. Discs of 1-cm diameter, containing counts/min each, were cut from the slab gel, dehydrated by immersion in Me2SO for 1 h, and immersed in the appropriate solution of PPO in MezSO for the times indicated. After incubation, Time of immersion in 15% w/w PPO (h) discs were dried and exposed to RP Royal X-Omat film at - 70 C for 48 h. The films were scanned with a Joyce- Loebl microdensitometer to measure the intensity of the fluorographic images. (A) Effect of PPO concentration. Discs were incubated for 3 h at 22 C for concentrations up to 30%, and at 37 C for concentrations above 30% in order to keep the PPO in solution. (B) Effect of incubation time in a 15% (w/w) solution of PPO in MezSO Table 1. Amounts of 3H, 14C and 35s required in a band to produce a visible image by jfuorography or autoradiography in a 24-h exposure Replica samples and serial dilutions of proteins labelled with [95S]rnethionine, [14C]valine, [3H]valine or [3H]proline (see Methods) were electrophoresed on slab gels 1.5-mm thick. After electrophoresis duplicate samples were processed for fluorography by the optimal procedure described in the text or dried for autoradiographv without PPO. A graded series of exposure times of the serially diluted samples was prepared to determine the minimum amounts of radioactivity which could be detected. Radioactivity in the bands under study was measured by scintillation counting of dissolved gel slices. Bands studied were 1 cm x 1 mm; 1 nci = 2220 dis./min Isotope Detection method Type of Film Radioactivity required for visible image after 24-h exposure 3H Autoradiography RP Royal X-Omat Kodirex Auto Process nci 1 >800 Fluorography with RP Royal X-Omat % (w/w) PPO Kodirex Auto Process 115 Blue Brand 18 Spraying dried gel twice with Omnispray RP Royal X-Omat > 221 ~~ -70 C -20 C +22 C > 800 > > s Autoradiography RP Royal X-Omat Kodirex Auto Process Fluorography with RP Royal X-Omat % (w/w) PPO Kodirex Auto Process C Autoradiography RP Royal X-Omat Fluorography with RP Royal X-Omat % (w/w) PPO a Values obtained by comparative microdensitometry, using the measured samples as standards. Eur. J. Biochem. 46 (1 974)

5 W. M. Bonner and R. A. Laskey 87 Artefactual blackening of the film sometimes occurs with gels containing MezSO, but this can be completely overcome by drying the gel first. MezSO itself is difficult to remove under vacuum (boiling point C); therefore it is replaced by water before drying. Soaking the PPO-impregnated gel in water leads to in situ precipitation of the PPO. This process turns the gel opaque, but surprisingly the fluorographic efficiency is not reduced and the gel can then be dried in one hour under vacuum (see Methods). Step 5. It is convenient to hold the film and the gel together between two glass plates secured with adhesive tape. The importance of the type of film and the temperature of exposure for fluorography are shown in Table 1 and have been discussed in detail by Randerath [2]. Our results on the effect of these two variables in fluorography of acrylamide gels (Table 1) confirm Randerath s [2] findings with fluorography of thin layers. RP Royal X-Omat film was found to be at least nine times more sensitive than the other films tested. Randerath [2] lists several possible alternative films for fluorography but in a later publication [7] Randerath and Randerath suggest use of RP Royal X-Omat. Exposure of RP Royal X-Omat film at -70 C rather than at -20 C or +22 C leads to 12-fold and 62-fold increases respectively in sensitivity for fluorography of 3H. For 35s and 1% fluorography, exposure at -70 C is approximately nine times more efficient than exposure at +22 C. If a -70 C freezer is not available, the film-gel sandwich can be buried in solid coz. Limits of Detection of 14C and 35s Although 35s and I4C can be detected in slab gels by autoradiography without PPO, Table 1 and Fig.4 show that these isotopes can be detected approximately ten times more efficiently with the fluorographic procedure presented in this paper. This finding contrasts with Randerath s observation [2] that 14C on thin-layer chromatograms can be detected equally efficiently by fluorography or autoradiography. This difference between thin layers and acrylamide gels could be explained if the absorption of /?-particles is greater in the dense matrix of a dried acrylamide gel than in a thin layer. This interpretation is supported by the observation that autoradiographic detection of 14C is much more sensitive (approximately 50-fold) in thin layers than in acrylamide gels (compare Table 1 and [2]). When the /?-particles are converted to light in situ by PPO this difference is greatly reduced. Thus the efficiency of direct autoradiographic detection of P-particles from 14C or 35s in polyacrylamide gels is so low that conversion of b-particles to light by PPO causes a ten-fold improvement, permitting the design Fig. 4. Comparison of fluorography and autoradiography for 3H or 14C-labelled proteins. Proteins were labelled with [3H]valine or [14C]valine in oocytes injected with EMC viral RNA counts/min of labelled proteins were electrophoresed in each slot of the gel. Duplicate slots were dried for autoradiography or processed for fluorography with PPO as described in the text. RP Royal X-Omat film was exposed to the gel strips at -70 C for 68 h of experiments with far smaller amounts of radioactivity. Limits of Detection of 3H Using the fluorographic procedure described here, 3000 dis. 3H/min in a band 1 x 0.1 cm will detectably blacken RP Royal X-Omat film in 24 h at -70 C. Bands containing 500 dis. 3H/min have been detected in, one-week exposures. Our attempts to detect 3H in gels by autoradiography without PPO (Table 1 and Fig.4) or by spraying dried gels with the commercial preparation Omnispray failed (Table 1). In polyacrylamide gels the detection of 3H by fluorography is almost as efficient as the detection of 14C or 35s by conventional autoradiography (Table 1 and Fig. 4).

6 88 W. M. Bonner and R. A. Laskey: Fluorographic Detection of 3H, 14C and 35S in Acrylamide Gels Consequently if equal numbers of measurable counts/ min (as distinct from dis./min) are compared a darker image is produced by fluorography of 3H than by autoradiography of I4C or 35s (Fig.4). This work was supported by the Medical Research Council and The Arthritis Foundation. We are grateful to Dr J. S. Knowland for valuable discussion, to Dr J. B. Gurdon for comments on the manuscript, to Mr A. D. Mills for excellent technical assistance and to Dr R. D. Barry and R. A. Lamb for making facilities available in the Division of Virology, Department of Pathology, Cambridge. REFERENCES 1. Wilson, A. T. (1958) Nature (Lond.) 182, Randerath, K. (1970) Anal. Biochem. 34, Laemmli, U. K. (1970) Nature (Lond.) 227, Laskey, R. A,, Gurdon, J. B. & Crawford, L. V. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, Peacock, A. C. & Dingman, C. W. (1968) Biochemistry, 7, Miller, L. & Knowland, J. (1970) J. Mol. Biol. 53, Randerath, K. & Randerath, E. (1973) J. Chromatogr. 82, W. M. Bonner s present address : Department of Health, Education and Welfare, National Institutes of *Health, 9000 Rockville Pike, Bethesda, Maryland, U.S.A R. A. Laskey, M. R. C. Laboratory of Molecular Biology, Postgraduate Medical School, University of Cambridge, Hills Road, Cambridge, Great Britain CB2 2QH