Ultramicrodetection of proteins in polyacrylamide gels

Ultramicrodetection Andrew IRBM,

Wallace Via Pontina

and

of Proteins Hans

Km 30.600,

Peter 00040

in Polyacrylamide

Gels

Saluz Porn&z,

Roma, Italy

Here we report the development of a highly sensitive procedure to detect proteins within separation matrices which should facilitate the characterization of rare proteins. The procedure is based on photochemical reactions where very low amounts of silver are deposited around proteins and in a series of steps are converted to silver sulfide. When this conversion is carried out in the presence of [“S]thiourea the resulting radioactive silver sulfide allows detection down to femtogram quantities of protein. In this work we applied the above principle to proteins separated on sodium dude& sulfate-polyacrylamide gels, thus not influencing physical and chemical parameters which are important for separation. This procedure should find application in any technique where detection of very low or limited amounts of proteins are required. 0 uw2 .40adelnio press. nno.

It is not an infrequent observation that protein factors which occur naturally at vary low concentrations play an important role, for instance, in the regulation of gene transcription. The characterization of such proteins prompts the development of increasingly sensitive protein purification and sequencing methods (blotting to suitable membranes, microbore LC, capillary electrophoresis, mass spectrometry, etc.). Nevertheless, all current purification methods rely on a sensitive procedure to monitor the quality of purification. So far a relatively high amount of precious sample had to be sacrificed to check the purity of the protein, since currently available detection procedures for purity testing are relatively insensitive. Naturally, antibody-based detection and affinity purification procedures can be used but, unfortunately, to raise antibodies specific to the protein of interest requires micro- to milligram amounts of the protein. Direct radiolabeling of proteins is a very sensitive method, but it changes their physical and often also their biological properties. The most sensitive detection procedure is indirect and involves silver staining of proteins after their separation within a matrix, such as a

polyacrylamide gel (1). Used in combination with Coomassie staining (2,3) or glutardialdehyde pretreatment (4) the detection limit we have observed (as described below) is approximately 4-40 ng of protein [although it has been reported that 10 pg could be detected under certain conditions, see Ref. (l)]. For the above reasons we sought to develop a reliable detection procedure which is ultrasensitive, uses readily available lab equipment, is straightforward, and could also be used in future microsequencing procedures to detect proteins blotted to suitable membranes. To this end we sought to enhance the sensitivity of existing silver-staining procedures by the use of radioactive isotopes. Useful hints were found in protocols employed in astro- and particle physics, radiological studies, and aerial photography where underexposed images were often enhanced by photochemical procedures (5-9). In a film emulsion containing silver halides, the image is formed by the selective reduction of irradiated silver particles after exposure to light or radiation followed by development and fixation. The developed silver in the autoradiogram was converted to a silver complex (silver thiosulfate) by means of Farmer’s reagent (10) and thence to radioactive silver sulfide by reaction with [%]thiourea (11) under alkaline conditions. Upon exposure of the treated autoradiogram to an X-ray film, an enhancement of the original image was produced. In contrast to the situation in a photograph, the image in a silver-stained gel is given by the higher deposition of silver around the protein bands compared with the gel matrix. This deposition might be due to a higher reducing potential of the gel region containing the biomolecules compared to its surrounding (1). The deposited silver can then be converted to silver bromide in a coupled reaction with potassium ferricyanide andpotsssium bromide to render the silver reactive with [%]thiourea with which it forms radioactive silver sulfide under alkaline conditions. In this way the separated proteins within the gel matrix are detected by autoradiography. The main advantage of employing radioactivity in a detection method is that the signal strength is

28

WALLACE

now time dependent, i.e., can be increased with exposure time. The sensitivity level of our new indirect labeling method is comparable to that of direct radioactive labeling procedures but does not interfere with protein separation. MATERIALS

AND

METHODS

Dichlorodimethylsilane was obtained from Sigma. Carbon tetrachloride (Ccl,), methanol (H&OH), ethanol (H,CCH,OH), acetic acid (H,CCOOH), silver nitrate AR (AgNO,), potassium carbonate AR (K&O,), 37% formaldehyde solution (stabilized with 10% methanol and Dolomite dust; HCHO), potassium ferricyanide AR (K,[Fe(CN),]), potassium bromide AR (KBr), sodium hydroxide AR (NaOH), 2-mercaptoethanol, and dithiothreitol (DTT)‘wereobtainedfromMerck. Radioactive [35S]thiourea (H,NCSNH,; sp act: 29.3 mCi/ mmol-supplied as dry powder; upon receipt, 1 ml of water was added and after dissolution, it was stored frozen at -20°C in loo-p1 aliquots) and Amplify were supplied by Amersham. For all solutions and washing steps deionized, double-distilled water (Mini-Q quality or equivalent) was used. Bovine serum albumin (BSA) was purchased from either Promega (blot qualified grade) or Pharmacia (gel filtration calibration kit). Other proteins were supplied by Pharmacia (gel filtration calibration kit). The yeast protein extract was a gift from Dr. A. Vitelli (IRBM, Rome, Italy). The protein samples to be stained were run on SDSpolyacrylamide gels (up to 9 cm x 5 cm x 0.7551.0 mm thick, with slot sizes of 5 X 1 mm) according to Laemmli (12). The detection method was developed using 10% polyacrylamide gels; however, good results were also obtained with lower (7.5%) or higher (up to 15%) acrylamide concentrations. All steps were carried out at room temperature while shaking the gels on a flat-plate shaker at 100 rpm. The gels were treated separately in polystyrene boxes of 17 X 25 X 4 cm (sufficient to hold 200-300 ml of solution). The protein samples were loaded with drawn-out coated glass microcapillaries (syringes were avoided). The samples were prepared in a total volume of 6 pl in sample buffers not containing DTT since this caused high background within the sample tracks (2.mercaptoethanol was used as a reducing agent instead). The gel glass plates were silanized with dichlorodimethylsilane (1% in carbon tetrachloride). The steps to detect the proteins within the gel matrix were carried out as follows. The gel was incubated in 40% methanol, 10% acetic acid solution for 1 h, using 250 ml of solution to fix the proteins. After removal of the fixing solution, the gel I Abbreviations used: DTT, dithmthreitol; bumin: SDS, sodium dodecyl sulfate.

BSA, bovine serum al-

AND

SALUZ

was soaked twice in 10% ethanol, 5% acetic acid for 5 min each time (250 ml). This was followed by two incubations in water for 5 min each time (250 ml). While these water wash steps were carried out, a 0.5 mg/liter [0.00005% (w/v)] solution of AgNO, was freshly prepared. After removal of the second water wash, the gel was incubated in the 0.5 mg AgNOJliter solution for 20 min (250 ml; the silver solution was added while shaking the gel). During this treatment, the following developing solution was freshly prepared: 0.01% (v/v) formaldehyde in 2% (w/v) potassium carbonate (for one gel, 250 ml of developing solution containing 5 g of potassium carbonate and 70 pl of 37% (v/v) formaldehyde dissolved in water was prepared). A small quantity (20-30 ml) of the developer or water was added while shaking the gel and removed after a few seconds, then the rest of the developing solution was added, and the treatment was carried out for 10 min (no visible signals at this stage). The developing solution was removed and the gel was washed in water four times, each step 5 min (250 ml). During these washing steps, 250 ml of a solution of 0.05% (w/v) potassium ferricyanide and 0.01% (w/v) potassium bromide in water was freshly prepared. After the last water wash step, the gel was incubated in the potassium ferricyanide/potassium bromide solution for 10 min (the solution was added while shaking the gel). To remove excess potassium ferricyanide/potassium bromide solution, the gel was washed four times in 250 ml of water, 5 min each time. During these washing steps, the [3”S]thiourea solution was freshly prepared 150 ~1 (50 pCi: 1.7 wmol; 0.13 mg) of the [35S]thiourea stock solution (1 mCi/ml of water) was added to 950 ~1 water and this was further diluted to 50 ml with 12.5 rn~ NaOH]. After the last water wash step, the water was removed and the gel incubated in the [35S]thiourea solution for 30 min. Finally the gel was washed three or four times in water for 5 min each step, then soaked for 30-40 min in 50 ml of Amplify solution with shaking (this latter step was facult&w), and then washed in water for up to 1 min. The treated gel was, without delay, dried on filter paper (Whatman 3) under VBCUU~ for 1 h at 80°C and exposed to an X-ray film which is sensitive to blue light (Kodak X-OMAT AR or equivalent) at ~30°C (with intensifying screens, if required) until the desired signals appeared upon film development. For any semiquantification, the film had to be linearized by p&lashing before exposure (14,15). The whole detection procedure is summarized in Table 1. The protein samples were prepared in lubrificated reaction tubes only (Multi Technology) and stocks of the commercially available proteins (see Materials and Methods) were prepared by weighing out 4 mg of each and dissolving this in 1 ml of water. These stocks were used to prepare the dilution series. Protein dilution series were performed either by using serial dilutions of a

ELECTROPHORETIC

Procedure

TABLE I for Radioactivation

of Protein

ULTRAMICRODETECTION

Bands

Treatment time (l-mm-thick gels) Ih 5 min 5 min 5 min

10% Ethanol, & acetic acid 10% Ethanol, 5% acetic acid Water Water 0.00005% AgNO, Rinse with developer or water 0.01% Formaldehyde in 2% &CO, Water water Water W&W

0.05% K,Fe(CN), KBr

5 min 20 min l-10 8 10 min 5 min 5 min 5 min 5 min 10 min

in 0.01%

Water

5 5 5 5

Water Water

water

50 pCi of [36S]thiourea in 50 ml of 12.5 rn~ NaOH Water (50 ml)

min min min mill

30 min 5 min 5 min 5 min

50 ml of

Amplify

30 min

solution

Rinse with water Dry the treated gels onto filter paper (Whatman 3) under YBC”“rn at 80°C Expose the dried gels to an Xray film which is sensitive to blue light (Kodak XOMAT AR or equivalent) at -80°C (with intensifying screen, if required) until the desired sienals mmar umm

1 min

Ih As required to visualize signals

defined protein mixture or by using coated glass microcapillaries with serial dilutions of only one protein. In the case of the yeast extract, the total protein concentration was estimated with the Bradford Coomassie dye-binding method (16) using a dilution series of bovine serum albumin, made from a stock solution as described above, to prepare the calibration curve. RESULTS

Determinationof

Silver

Nitrate Concentration

The previously described sorbed layer of allylthiourea lows the reaction: H,N-RC=S

reaction and silver

+ 2Ag+ + 20H-

between bromide

an ad(17) fol-

-w A&

+ RC=N

+ 2H,O.

OF PROTEINS

29

In a similar manner it is to be expected that silver bromide reacts with [YS]thiourea to form insoluble radioactive silver sulfide, thus indicating that for conversion of silver to silver sulfide 1 mol of thiourea is required for 2 mol of silver. When we attempted to directly react silver nitrate-treated gels with [V]thiourea, a high general background was obtained over the whole gel and only large amounts (400 ng or more) of proteins gave bands which were visible above the background (data not shown). This seems to be understandable, if it is assumed that after soaking the gel (10% acrylamide) in a solution of silver nitrate to equilibrium, the silver nitrate distributes throughout the gel and reacts with thiourea to give a uniform deposit of insoluble silver sulfide within the entire gel, which cannot be removed prior to autoradiography. Since this direct approach did not work as expected, the procedure of Ansorge (13) was used to stain the protein bands with metallic silver deposits. In this procedure, silver ions in the vicinity of the protein are reduced to metallic silver by reaction with formaldehyde at a faster rate than in the remainder of the gel, due to the fact that the protein acts as a catalyst in this reduction reaction. The excess nonreduced silver nitrate in the bulk of the gel can be washed away. In order to render the deposited silver reactive with thiourea, the gels were treated with a potassium ferricyanide-potassium bromide bleach bath (18,19) to form silver bromide, which was then converted to silver sulfide with radioactive thiourea. Although this led to an improvement over the direct method, the results were not acceptable in that small quantities of proteins could still not be seen above the general high background (data not shown). It seemed likely that the amount of radioactive thiourea used in the experiment was many-fold lower than the amount of silver remaining in the gel and thus insufficient to obtain a signal to background ratio large enough to visualize small amounts of proteins. Using a 5.9 mM silver nitrate solution as in conventional silver staining (13), the maximum concentration of silver nitrate in the 10% gel will be approx 5.3 nm. This large excess of silver nitrate would require about 1.6 mm01 (49 mCi) thiourea for complete conversion to silver sulfide. Rather than increase the amount of radioactive thiourea we decided it would be simpler to decrease the amount of silver nitrate to a level around that of the thiourea (50 pCi [35S]thiourea corresponded to 8.5 PM thiourea at the specific activity used). A convenient concentration was found to be 2.9 PM silver nitrate, i.e., 0.125 mg per 250 ml of water (volume used for gels up to 9 cm X 5 cm X 0.75-1.0 mm thick, with slot sizes of 5 X 1 mm) and approximately 20 @g of total proteins per gel. This silver nitrate concentration is at least 2000 times less than that used in silver-staining procedures (13) and development of the silver at this point does not result in visible signals.

30

FIG. 1. Effect BSA amounts g/liter-KBr, g/liter-KBr, tions resulted

Radioactiuation

WALLACE

of different were loaded: 0.01 g/liter: 1 g/liter; (E) in a decreasing

AND

SALUZ

bleach bath concentrations on the protein-sdver radioactivation procedure. For each panel 50 ng at the left, 100 ng at the right. The following inereasrng concentrations were used: (BJ K,Fe(CN),, 0.5 g/liter-KBr. 0.05 g/liter: (C) K,Fe(CN),, 1 g/bter-KBr, 0.1 g/liter; K,Fe(CN),, 20 g/liter-KBr, 2 p/liter; (F) K,Fe(CN),. 40 g/Ilk-KBr, 4 g/liter. Increasingbleach signal strength and very faint bands became invisible. The exposure time was 10 h.

of Siluer

Deposited

on Proteins

In order to determine which reagents other than silver influenced the signal-to-background ratio most strongly, titrations were performed. This not only revealed the importance of each compound but also the optimal concentration range and tolerance over this range. Knowing from classical photochemistry (18-201 that the bleach bath composition may influence the intensity of the final photographic image, initial titrations concerned the bleach bath concentration. The effect of different bleach bath concentrations on the proteinsilver radioactivation procedure is demonstrated in a few examples in Fig. 1. The optimal concentration range was found to be between 100 and 1000 times (Figs. 1A and 1B: K,[Fe(CN),], 0.1-0.01 g/liter and KBr, O& 0.05 g/liter) below the values used in photography (K,[Fe(CN),], 50 g/liter and KBr, 10 g/liter). In addition, as mentioned above, another important factor concerns the total amount of [%]thiourea used. An excess of thiourea over the silver bromide is required to avoid the possibility that the lower silver bromide-density e.reas are disproportionally more radioactivated than the higher density areas. Some results of using different [%]thiourea concentrations are shown in Fig. 2. For low protein amounts (up to a few nanograms) 170340 nmol (5-10 pCi) of [%]thiourea in B reaction volume of 50 ml was sufficient. However, knowing that in a

typical experiment several might be loaded onto a gel, the above protein amount, pCi: 0.13 mg) [%]thiourea to be sufficient for all protein periments. Factors

which

InfEuence

(A-F) two different (A) K,Fe(CN),, 0.1 (D) K,Fe(CN),, 10 bath concentra.

different protein samples thus exceeding drastically we employed 1.7 pm01 (50 per reaction, which proved amounts used in our ex-

the Signal-Background

One of the most important fluencing the signal-background

Ratio

parameters directly inratio is the washing

FIG. 2. Titration of optimal [%]thiourea concentmtmns (sr, act: 29.3 mCi/mmol). The gels were exposed for 16 h: CA) 30 ,Ci, ,B, 50 rCi, and (0 70 rCi. Amounts of radmactivity greater than 50-70 &i did not result in an improved signal for all the different protein quantitiea described in the text.

ELECTROPHORETIC

ULTRAMICRODETECTION

step after bleach treatment and after soaking the gel with thiourea since potassium ferricyanide, a component of the bleach bath, reacts with thiourea. Therefore the water-washing steps following the bleach bath treatment should he sufficient to remove excess reagent. It is recommended that the gel is washed while shaking (100 rpm) for 4 X 5 min. Washing the gel after radioactive thiourea treatment is essential as contamination of the gel with unused reagent will increase the background signal to an unacceptable level. Since thiourea is easily soluble in water the absolute time is not critical; however, at least two to three exchanges of water should be used to wash the gel properly. A too extensive wash is not harmful due to the insolubility of silver sulfide under these conditions. Good results were obtained by washing the gels three times in 50 ml water and 5 min each wash. Certain compounds which are normally innocuous in conventional detection procedures may take part in the radioactivation process and thus drastically increase the background. For instance, the proteins are usually reduced in a sample buffer containing DTT to resolve them into their constituent subunits upon SDS-polyacrylamide gel electrophoresis. However, this treatment produced smears within the gel tracks upon radioactivation. The use of Z-mercaptoethanol as a substitute for DTT solved this problem. The use of glutardialdehyde as a fixing agent of small peptides in combination with glycine-containing running buffer may cause an increase in the overall background. If such a combination should be used, a very intensive washing of the gels before silver nitrate treatment is recommended to remove the aldehyde trapped in the gel matrix. To avoid superimposition of the image from parts of one gel on another, each gel should be treated in a separate container. Finally it was observed that the most regular staining was obtained when all reagent solutions were prepared freshly and the gels incubated with these solutions while shaking (100 rpm) on a flat-bed shaker. To receive overall strong signals, the gels may be incubated in a small volume of Amplify (Amersham) solution, which is a fluorographic enhancement reagent (this is recommended whenever small amounts of proteins are used). This treatment resulted in a conversion of the p-particle energy to light energy in a wavelength region where X-ray films are more sensitive when exposed at -70 to -80°C. If a preflashed film (0.2 OD) was used (14,15), a protein band (5 mm gel slot) containing 0.37 Bq (0.01 nCi) was visible in approximately 500 h. Within the linear range of the film, the strength of the radioactive signal is directly correlated to the exposure time (Amersham Amplify instruction manual). By using one or two intensifying screens (21) and/or baking the films in an 6% “forming gas” (8% H, + 92% N,)

OF PROTEINS

environment (22-24) time can be obtained. Sensitivity

31

further

Comparison

reduction

between

Different

of the exposure

Detection

We were naturally interested to compare the sensitivity of this procedure with commonly used staining methods, such as Coomassie brilliant blue, silver staining, and a combination of both. The results are shown in Fig. 3. All the different steps were optimized using BSA (M, 67000) and ovalbumin (M, 43000) as standard proteins. The highest amounts of BSA and ovalbumin used for titrations were 400 ng and the lowest 400 fg. These protein amounts were determined according to weight and these numbers may be, if anything, an overestimate of the amounts effectively present, since salts or traces of other proteins could still contaminate the sample. The lowest detected amount of these proteins with Coomassie brilliant blue staining (Fig. 3a) was 40 ng. With the more sensitive silver-staining method (Fig. 3b), between 4 and 40 ng could be detected. A combination of Coomassie brilliant blue and silver staining (Fig. 3c), led to the detection of amounts down to 4 ng but with a higher signal intensity. The radioactivation procedure involving [35S]thiourea (Fig. 3d) allowed the detection of amounts down to approximately 400 fg. It can be seen from Fig. 3d that the signal response is not linear, i.e., in the example shown it can be seen that small amounts of proteins show almost the same signal intensity, whereas large amounts give relatively small signals when compared with procedures involving silver staining (Figs. 3b and 3~). Similar observations were made by Owunwanne et al. (6), where radioactivation of weak bands on autoradiograms resulted in a nonlinear response upon reexposure to a fresh X-ray film (6). Resolution

and Detectability

upon

Radioactivation

of a

To demonstrate that this method is not only suited for a few selected proteins, i.e., BSA and ovalbumin, but can be applied generally, we radioactivated proteins from a whole yeast extract and compared the results with those from Coomassie blue- and Coomassie plus silver-staining experiments (Fig. 4). The purpose of this experiment was to see whether the bands in the radioactivated gel stained in a manner comparable to the other procedures. Thus we were not looking for a direct comparison on the basis of sensitivity in this experiment. The proteins of the yeast extract (4.2 pg total protein per lane) were separated on a 7.5% SDS-polyacrylamide gel as described above. Similar to Fig. 3d, the response of the radioactivated proteins (Fig. 4, lane 3) is not linear. Protein bands staining with a variety of weak intensities in the Coomassie plus silver track (Fig. 4, lane 2) give almost equal responses in the radioacti-

32

WALLACE

AND

SALUZ

FIG. 3. Sensitivity comparison between dafferent detection procedures on 10% l-mm-thick SDS gels with slot sizes of 6 m m wdth. (a) Coomassie blue staining. (b) Sdver staining. cc) Caamassie and silver staining. cd) Radioactivatmn procedure involving [%]thiourea, exposed for 3 weeks. Lanes I-7: 400 ng, 40 ng, 4 ng. 400 pg, 40 pg. 4 pg, 400 fg of each protein, i e., bovine serum albumin (B) and ovalbumm (0). Caomassie blue staining (a) detected down to 40 ng of each protein. silver staining (b) down to 4 ng in the best case, Coomassie plus silver staining (c) dawn to 4 ng but with higher signal Intensity, and with radioactivation (dJ down to 400 fg.

vated track (Fig. 4, lane 3). When a protein mixture as complex as that present in yeast is separated on a minigel, the background within the sample lanes will in-

crease in proportion to the sensitivity of the detection method used. When applying less complex and less concentrated samples this effect could not be observed (see Fig. 3d). In the example of Fig. 4, the same total amount of protein was loaded in all lanes; the exposure time of the gel in lane 3 was chosen to give band intensities similar to those of the other methods and thus allows a direct comparison of the results. The radioactivation technique produces band patterns comparable to the other procedures, even when detecting such high amounts of proteins. However, it is clearly different concerning sensitivity in that the signal response may vary less widely over a broad range of different protein amounts. DISCUSSION

FIG. 4. Comparison of the resolutmn and detectability of proteins in a sample of yeast extract between different sensitive protein staining procedures on 7.5%. l-mm-thick SDS gels with slot sizes of6 m m width. In each track shown, the total amount of protein (determmed by the Bradford assay as described under Materials and Methods) loaded was 4.2 pg. The tracks shown were obtained as follows: Staining with Coomassie brilliant blue (1). Coomassie plus silver staining ?2), and radioactivation with an overnight exposure (3). The background in I is slightly greyer than that in the other panels due to the photographic exposure required to reveal the fainter Coomaasiestamed bands.

In this paper we describe the development of an ultrasensitive protein detection procedure which should be of great assistance in the purification of rare proteins. In addition it could be applied to peptide mapping, forthcoming sequencing techniques, and estimation of small amounts of proteins on SDS-PAGE. The latter is possible since exposure on a linearized X-ray film allows a correlation between the amount of signal obtained and therefore a semiquantification of the material present. With small modifications it should also be applicable to

ELECTROPHORETIC

ULTRAMICRODETECTION

the detection of other h&polymers, such as nucleic acids (25), etc. In contrast to conventional staining procedures where a directly visible signal is produced, in the technique described here, we have introduced the use of ra dioisotopes since the detection of radiolabeled material is time dependent, thus allowing the detection of much smaller quantities than previously possible. Several isotopes, such as iodine-125, sulfur-35, promethium-147, cadmium-115m, iron-55, nickel-63, mercury-203, silverllOm, gold-195, polonium-210, uranium-233, americium-241, or californium-252 (7), would be well suited for very sensitive enhancement procedures but due to the lack of appropriate equipment to ensure the safe handling of most of the above isotopes in general biochemical laboratories only two were of interest: iodine and sulfur. Iodine-125 due to its high emission energy would allow rapid detection hut with the consequence of strong scattering. The scattering of the weak @-emitter “S is much less strong, thus guaranteeing sharp signals when exposed to an X-ray film. One possible way to indirectly label proteins separated in gels with the above isotope was to convert the deposits of metallic silver obtained at the sites of protein in conventional silver staining to radioactive silver sulfide by means of [%]thiourea, because similar reactions were already applied in photochemistry and physics (lo), for example in the archiving of rare photographs (sulfur toning). The first step of the procedure is the “staining” of the gel with silver nitrate, which results in the diffusion of the silver salt throughout the gel matrix. During this process, silver ions tend to associate with the trapped protein, which results in the concentration of silver around the protein bands being higher than in the gel matrix. The second step is to react silver nitrate with formaldehyde under basic conditions to produce metallic silver, a process called development. Proteins act as catalysts in this reaction, leading to a faster deposition of silver metal in the area of the protein bands, compared to that in the rest of the gel. The nonreduced silver, which is the source of high background in the later steps must be washed away at this stage. Here we used formaldehyde as developer, as is also applied in biochemical silver-staining procedures (10,13). Other developers that produce an even finer grain, for example, 1.phenyl-3.pyrazolidone [phenidone; Refs. (26,2’7)], were not tested since much longer developing times are required, but their use may well result in a further increase of the final staining quality. In a subsequent step known as bleaching, using potassium ferricyanide and potassium bromide, the deposited silver is converted to AgBr. The silver bromide is then transformed to silver sulfide by reaction with thiourea (known as sulfur ton ing). In our staining procedure, when used with [%]thiourea, the result is a deposit of radioactive silver sulfide at the sites of proteins.

33

OF PROTEINS

For performing all of our experiments we used relatively thick gels (1 mm) and wide slots (5 mm). Consequently the overall sensitivity could be even further increased when ultrathin gels with small slots are used. The high sensitivity achieved with our method may involve the handling of minute amounts of protein and thus some care must he exercised to avoid inadvertent sample contamination or loss due to unspecific binding of the protein to equipment surfaces. In fact, we would advise that the sensitivity of the method should not be exploited to a point where accurate handling of the sample can no longer be ensured. In conclusion, we mention some of the many possible applications involving this method. An important aspect is the ability of this method to detect certain types of synthetic peptides which conventional procedures fail to stain (A. Pessi, personal communication). This may be due to the fact that the few silver grains which form around these peptides are not sufficient to be visualized directly but can be usedto produce radioactive signals which can he revealed by autoradiography or phosphorimaging upon adequate exposure time. Finally, an application which exploits the high sensitivity to its full potential may be in fields where samples are limited to very small amounts and have a unique origin, such as forensic science, archeology, or palaeontology. ACKNOWLEDGMENTS Dr. Karin Wiebauer (IRBM, Rome, Italy) deserves our thanks for critical proofreading of the manuscript and the resulting excellent suggestions. We are grateful to Dr. Rudolf Gschwind (Department of Physics, University of Bale, Switzerland) and Professor Riccardo Car&e and Dr. Josef Jiricnv (both at the IRBMl for their h&ful discussions and their great i&est in this work. We alao thank’& Alissandra Vitelli (IRBM) for the kind gift of the yeast extract. REFERENCES 1. Merril,

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