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EXPERIMENTAL PATHOLOGY AND PARASITOLOGY, 6/13, 2003 Bulgarian Academy of Sciences An improved procedure for silver staining of protein bands on polyacrylamide gels S. ZACHARIEVA1, E. PANEVA2, J. YANEVA2 1 Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria 2 Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Abstract A streamlined protocol that allows high sensitivity visualization of protein bands by silver staining is reported. The entire process, from gel to record, can be accomplished within three hours. Comparing with the classical Coomassie brilliant blue dying of proteins, the proposed silver staining procedure is faster, clearer, more sensitive and easily performed. It allows detecting protein bands in polyacrylamide gels within the range of 10 ng/mm2. The stain is applicable to various acrylamide gel thickness and gel concentrations. Key words: histone H1, polyacrylamide electrophoresis, proteins, silver stain. Introduction Silver stain technology was first applied for histochemical purposes; ammoniacal silver complexes have been used in histology for a long time to obtain fine definition unobtainable by other methods. Later on the approach was applied also for detection of proteins and nucleic acids on polyacrylamide gels (S w i t z e r et al., 1979). Especially for proteins, the original procedure was modified and improved repeatedly and it was firmly demonstrated to be faster and more sensitive (M e r r i l et al., 1981; M o r r i s s e y, 1981; S a m m o n s et al., 1981; M o l d et al., 1983; N i e l s e n and B r o w n, 1984; W i l l o u g h b y and L a m b e r t, 1983; W r a y, 1983; K u r o s a k i et al., 1984; N i e l s e n and B r o w n , 1984; B l u m et al., 1987; H i l b e r t et al., 1995) than the classical Coomassie brilliant blue staining (F a i r b a n k s et al., 1971). Nowadays many investigators examine protein samples only in extremely limited amounts, i.e. during the process of isolation and purification of individual fractions. That is whay it is so important to utilize staining with maximal sensitivity and quickness. Recently we have described a simplified protocol for DNA silver staining in polyacrylamide gels (P a n e v a et al., 2000). Here we proposed an adapted version for imaging of protein bands on polyacrylamide gels by silver staining: an example with major nuclear protein histone H1 and various protein markers. The procedure is fast, easy to handle and effective for testing huge series of fractions during the process of protein isolation and purification. 35 Materials and Methods Chemicals. All chemicals acrylamide, N, N-methylene-bisacrylamide (bis), sodium dodecylsulphate (SDS), ammonium persulphate (APS), tetramethylenediamine (TEMED), Tris (hydroxymethylaminomethane), glycine, BPB (bromphenol blue), Coomassie brilliant blue R-250, silver nitrate and formaldehyde were of analytical grade and purchased from Sigma (Saint Louis, MO, USA). Isolation and purification of histone H1. The major nuclear protein histone H1 was purified from mouse liver nuclei under non-denaturing conditions on CM Sephadex C25 columns following the procedure described by B a n c h e v et al., 1991. The concentration of histone H1 was determinated spectrophotometrically using an extinction coefficient of 1.85 ml. cm-1. mg-1 at 230 nm wave length. Polyacrylamide electrophoresis under denatured conditions. Electrophoresis was performed in vertical polyacrylamide slab gels (150 × 120 × 1 mm), containing 0.1% SDS (L a e m m l i, 1970; S a m b r o o k and R u s s e l, 2001). About 20 mm long was the 6% stacking gel on the top of the 15 % separating gel (acrylamide to bisacrylamide ratio 29:1), containing 4.5% Tris-HCl (pH 8.8), 0.1% SDS, 0.025% ammonium persulphate, and 0.05% TEMED. PAGE running buffer (pH 8.3), contained 0.3% Tris, 1.44% glycine and 0.1% SDS. Usually the gels were first prerun for 30 min at 300V (constant) and 60 mA and then run with protein samples for about two hours at 250 V and 50mA. All the time plastic gloves were worn while handling the gels. Sample preparation. The protein samples (different amounts of histone H1 or various marker proteins) were prepared in sample buffer (pH 8.0) containing 5% glycerol, 0.121 % Tris, 1% SDS and 0.0292% EDTA. The samples were heated in water bath at 95oC for 3 min and allowed to cool at room temperature. Then 0.1% bromp36 henol blue as a tracking dye was added to each sample and they were layered into gel slots using special loading tips. When the tracking dye reached about 2 cm of the bottom of the gel, the current was turned off and the gel was removed. Coomassie blue R-250 staining. Two grams of Coomassie blue R-250 reagent (Sigma, MO, USA; # B-0149, Lot 13H5002) were dissolved in 400 ml of methanol; then 300 ml of deionized water and 200 ml of 50% acetic acid were added with continuing stirring to complete the preparation (0.2% Coomassie brilliant blue R-250 in 40% methanol 10% acetic acid). For Coomassie staining of one of the two identical gels was placed in 150 ml of this solution for four hours under slight shaking. Then the gel was unstained with 10% methanol 5% acetic acid with many changes until the background became clear and the protein bands apparently visible. Silver staining of protein bands. The whole protocol of the silver stain procedures with determinated standard conditions is presented in Table 1. The stock solution of silver nitrate (Sigma, USA, catalog number S-8157) has been prepared with deionized water and kept in dark bottles at 4oC and used within 1-2 weeks. Developer solution formaldehyde (cat.# F-1635) in sodium carbonate has been prepared always just before use. Protein silver-staining procedure. The pictures of the stained gels were taken with PRACTIKA MTL5 camera (objectglass 2.8/50) using back white light source. When needed the gels were died between two wet cellophane sheets at room temperature or in vacuum dryer and kept as files for a long time. Results All single steps of the silver staining procedure for detection of histone H1 bands after polyacrylamide electrophoresis are outlined in Table 1. The propo- Table1. Main steps of the proposed protein silver-staining procedure STEP DURATION AND TEMPERATURE 1. Fixing the gel in 50% methanol10% acetic acid. 2. Thorough washing with dH2O 3. Fixation the gel in 1% nitric acid 4. Rinsing twice with dH2O. 5. Soaking for impregnation in 0.3%AgNO3. 6. Rinsing briefly with dH2O 7. Developing in 2.5% NaCO3 + 75ìl 37% formaldehyde in 100ml dH2O prepared just before use. 8. Rinsing twice with dH2O. 9. Taking a picture using back illumination of white light. 10. Fixation in 20% methanol5 % glycerol 11. Rinsing with H2O. 12. Drying the gel between two wet cellophan sheets at 13. Keeping as a record. sed procedure represents a compilation of our previous and other authors protocols (S w i t z e r et al., 1979; M e r r i l et al., 1981; M o r r i s s e y, 1981; S a m m o n s et al., 1981; M o l d et al., 1983; N i e l s e n and B r o w n, 1984; W r a y, 1983; K u r o s a k i et al., 1984; N i e l s e n and B r o w n, 1984; B l u m et al., 1987; H i l b e r t et al., 1995; P a n e v a et al., 2000). Since 1990 we have been investigated the interaction of the linker histones (H1, H5, H1° and cognates) with DNA (Y a n e v a et al., 1990; Y a n e v a and Z l a t a n o v a, 1991; Y a n e v a et al., 1995; Z l a t a n o v a et al., 1995; Y a n e v a et al., 1997). Many observations point to the involvement of linker histones (H1, its subtypes and variants) in the switching mechanisms that determine gene expression (B u t t i n e l l i et al., 1999). The importance of these investigations often forced us to isolate, purify and analyze simultaneously variuos types of linker histones from different mammalian tissues. For direct comparison two identical NOTES 2 hours, r.t. up to 4 hours 3 times, 2 min each 20 min 1-2 min each 20 min, r.t. slight shaking 1-2 min, r.t. 10-12 min up to appearing of brownish bands 1-2 min each 15-20 sec 1-2 h, r.t. 2-3 min o.n., 23-26oC up to 48 hours forever polyacrylamide gels with decreasing amounts of histone H1 were stained either with silver stain and Coomassie brilliant blue stain (fig. 1). It is well visible that the protein bands on silver stained gel (fig. 1, A) are quite more prominent than the same on the identical Coomassie brilliant blue stained gel (fig. 1, B). On the first gel the bands are apparently visible up to lane 8 (50ng of the protein sample), but on the second gel up to the forth (3ìg sample). The same protocol for silver staining was applied on another protein samples various protein markers (Sigma, USA) with different molecular weights (fig. 2). We have avoided an overnight fixing in methanol-acetic acid because separated experiments with isotope labelled proteins showed decreasing of the silver stain sensitivity by 20% (unpublished observations). Sometimes several portions of methanol-acetic acid were useful for initial fixing of the separated proteins on PAAG. The consequence was a satisfactory reduction in background. 37 À Â 1 1 2 2 3 4 3 4 5 5 6 7 6 7 8 Fig. 1. Silver staining of the major nuclear protein histone H1 on PAAG (A) compared with Coomassie - stained one (B). Decreasing amounts in micrograms of purified mouse liver histone H1 were loaded onto similar lanes of two identical 15% polyacrylamide gels containing 0.1% SDS: 10ìg (lane 1); 5ìg (2); 4ìg (3); 3ìg (4); 2ìg (5); 1ìg (6) ; 0.5ìg (7); 50ng (8). Note that the protein bands on the gel obtained after silver stain are visible up to lane 8 (50ng) while after Coomassie blue stain only up to lane 4 (3 ìg) 38 phosphorylase (rabbit muscle) - 97.4 KD bovine serum albumin - 66.0 KD albumin (chicken egg) - 45.9 KD carbonic anhydrase - 29.0 KD β - lactoglobulin - 18.4 KD top bottom a - lactoglobulin - 14.2 KD histone H1AB histone H1ο Fig. 2. Another example with silver staining mixture of various marker proteins with different molecular weights -500 ng of each sample (denoted above) and histone H1 subfractions obtained after 12% polyacrylamide electrophoresis (according to Laemmli, 1970) Discussion The exact mechanism of the silver stain reaction is still unclear. Probably it involves the formation of initial nucleation sites at certain charged amino acid side chains of the proteins, followed by the rapid buildup of silver deposits in the presence of a reducing agent (usually formaldehyde) from free silver ions in the gel or in solution. The deposition of silver ions is allowed to continue until protein bands become visible and the background is unacceptable (M o l d et al., 1983; K u r o s a k i et al., 1984; H i l b e r t et al., 1995). The main drawbacks of most protocols for protein silver staining have been the long duration and an unspecific background staining. Here we present a quick and quite sensitive procedure for imaging of protein bands by silver stain avoiding that drawbacks and obtaining a transparent background. The process might be completed effectively within three hours. This prescription allows to detect proteins in PAAG within the range of 10 ng/mm2. It should be mentioned that the proposed procedure is especially suitable for the cases of fast testing of the column fractions during protein isolation and purification. Abbreviations used: AA, acrylamide; APS, ammonium persulphate; KD, kiloDaltons; o.n., over night; PAAG, polyacrylamide gel; r. t., room temperature; SDS, sodium dodecyl sulphate; TEMED, tetramethylenediamine; Tris hydroxymethylaminomethane 39 Acknowledgements This work was supported by a research grant (K1003/00 to J.Y.) from the Bulgarian Ministry of Education and Science. The excellent photo service of Mr. Z. Apostolov during the experiments is also appreciated. References B a n c h e v, T., L. S r e b r e v a, J. Z l a t a n o v a. Purification of histones H10 and its subfractions under non-denaturing conditions. Biochim. Biophys. Acta, 1073, 1991, 230-232. B l u m, H., H. B e i e r, H. J. G r o s s. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis, 8, 1987, 93-99. B u t t i n e l l i, M., G. P a n e t t a, D. R h o d e s, A. T r a v e r s. The role of histone H1 in chromatin condensation and transcriptional repression. In: E. M. 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