A simple modification of Blum's silver stain method allows for 30 minute detection of proteins in polyacrylamide gels. Michael V. Nesterenko *, Michael Tilley, ...
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J. Biochem. Biophys. Methods 28 (1994) 239-242
biochemicaland biophysical methods
Short Note A simple modification of Blum's silver stain method allows for 30 minute detection of proteins in polyacrylamide gels Michael V. Nesterenko *, Michael Tilley, Steve J. U p t o n Division of Biology, Ackert Hall, Kansas State University, Manhattan, Kansas 66506, USA
(Received 5 November 1993; accepted 23 December 1993)
Abstract
A simple and rapid protocol for silver staining of proteins following electrophoresis in polyacrylamide gels (PAGE) is described. We have reduced the number of steps in the procedure of Blum et al. (Electrophoresis (1987) 8, 93-99), and shortened fixation and washing times so that efficient detection of proteins can be achieved within 30 min. In common with more time-consuming silver-staining methods, the present protocol is capable of detecting nanogram quantities of proteins on a colorless background and is suitable for rapid screening of large numbers of samples. Key words: Polyacrylamide gel electrophoresis; Silver stain; Fast protein fixation
1. Introduction
A variety of techniques have been developed for the sensitive silver staining of biopolymers following PAGE [1-5]. However, most silver stain protocols employ lengthy fixation times and repetitive washes. Although fixation prevents diffusion of separated proteins and removes interfering substances such as sodium dodecyl sulfate (SDS), Tris buffers, and other salts [6], it increases the amount of time needed to visualize results. Commercially available silver-staining kits for rapid (40-60 min) visualization of proteins in polyacrylamide gels rely on elevated temperatures a n d / o r a special development chamber [7]. This note describes a silver-staining procedure based on the methods of Blum et al. [8] and Heukeshoven
* Corresponding author. Fax: + 1 (913) 5326653. 0165-022X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-022X(94)00002-U
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Table 1 Procedure for 30-min silver-staining method
a
Steps
Solution
Time
1 2 3 4 5 6 7 8 9 10
Fixation Rinse Wash Rinse Pretreat Pretreat Rinse Impregnate Rinse Develop c
5 min 3×5 s 5 min 3x5 s 5 min 1 min 3x5 s 8 rain 2×5 s 10-20 s
11 12
Stop Rinsed
60 ml acetone stock b; 1.5 ml TCA stock b; 25/zl 37% HCHO ddH20 ddH20 ddH20 60 ml acetone stock b 100/~l Na2S203 .5H20 stock b in 60 ml ddH20 ddH20 0.8 ml AgNO 3 stock b; 0.6 ml 37% HCHO; 60 ml ddH20 ddH20 1.2 g Na2CO3; 25 ~137% HCHO; 25/xl Na2S203.5H20 stock a; 60 ml ddH20 1% glacial acetic acid in ddH20 ddH20
30 s 10 s
a All steps were performed in glass or plastic containers on a shaker at room temperature (approx. 23°C). The volumes of all solutions were 60 ml for mini-gels 0.75 mm thick. b Stock solutions: 50% acetone in ddH20; 50% TCA in ddH20; 20% AgNO 3 in ddH20 (store in dark; shelf-life up to 4 months); 10% NaES203 .5H20 in ddH20 (shelf-life about 4 months). c A brown precipitate may appear upon contact of the gel with the developer. It can be dissolved by vigorous shaking.
and Dernick [7,9], but requires only about 30 min for completion by employing rapid fixation and washing steps. In addition, the protocol is used at room temperature without the need of special equipment.
2. Materials and methods
All chemicals used in SDS-PAGE, including molecular-mass markers (MWSDS-70L and MW-SDS-200), were obtained from Sigma Chemical (USA). Silver nitrate, formaldehyde, anhydrous sodium carbonate and sodium thiosulfate were purchased from Fisher Chemical (USA). Protein standards were prepared by SDS-PAGE on a BioRad Protean II mini-electrophoresis unit according to Laemmli [10] in 0.75 mm thick slab gels containing 13.4% acrylamide and a low concentration of N,N'-bisacrylamide (ratio of crosslinker to total monomers 0.62%). This acrylamide/bisacrylamide ratio has been shown previously to offer better resolution of high-molecular-mass markers and allows all proteins from 10-200 kDa to be resolved in a single non-gradient gel [11]. The silver-staining procedure is presented in Table 1.
3. Results and discussion
All silver-staining methods are initiated by treatment of the gel with different fixatives for 20 min to overnight [12]. In the present protocol, the short (5 min)
M. I~. Nesterenko et al. / J. Biochem. Biophys. Methods 28 (1994) 239-242
1
2
3
4
5
6
7
8
9
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10
Fig. 1.30 min silver stain of proteins separated by SDS-PAGE. Low-molecular-mass markers, lanes 2, 4, 6, 8, 10; high-molecular-mass markers, lanes 1, 3, 5, 7, 9. Sizes of markers (in kDa) shown on the left. All markers were stained as described in Table 1. Lanes 1 and 2, 11 ng total protein each; lanes 3 and 4, 33 ng total protein each; lanes 5 and 6, 100 ng total protein each; lanes 7 and 8, 300 ng total protein each; lanes 9 and 10, 900 ng total protein each.
fixation in 50% acetone, 1% trichloroacetic acid (TCA) and 0.015% formaldehyde (HCHO) with subsequent washing with double-distilled water (ddH20) (steps 1-4, Table 1) prevents the diffusion of separated proteins and removes substances which interfere with the staining from the gel. After a second incubation in 50% acetone (step 5, Table 1), the gel is easily impregnated with the silver-staining agent. The impregnation of the gel with silver nitrate is the basic step and the level of sensitivity relies, in part, on the level of background staining [12]. However, the use of sodium thiosulfate in the pretreatment and developing solution prevents surface staining and results in a clear background [8]. A typical fast silver-stained gel pattern of proteins is shown in Fig. 1. The sensitivity of this 30 min procedure is comparable to original, more lengthy methods [7,9] and can be used for staining proteins with a broad range of molecular sizes. After stopping staining and rinsing in ddH20, the gel was incubated for 15 min in 1% glycerol and dried under vacuum between two cellophane sheets. The image of the dried gel is stable. In our laboratory, this silver-staining method is used for detection of proteins in polyacrylamide gels following isoelectric focusing, native and denaturing electrophoresis, and is used for gels of different compositions and thicknesses ranging from 0.4-2.0 mm. In addition, the present method makes it suitable for rapid electrophoresis analysis of large numbers of protein samples.
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4. Acknowledgements This research was supported by NIH grant AI30881 to SJU. This is Kansas Agricultural Experiment Station contribution No. 94-70-J.
5. References [1] Oakley, B.R., Kirseh, D.R. and Morris, N.R. (1980) A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105, 361-363. [2] Morrissey, J.H. (1981) Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117, 307-310. [3] Ohsawa, K. and Ebata, N. (1983) Silver stain for detecting 10-femtogram quantities of protein after polyacrylamide gel electrophoresis. Anal. Biochem. 135, 409-415. [4] Merril, C.R., Harrington, M. and Alley, V. (1984) A photodevelopment silver stain for the rapid visualization of proteins separated on polyacrylamide gels. Electrophoresis 5, 289-297. [5] Vesterberg, O. and Gramstrup-Christensen, B. (1984) Sensitive silver staining of proteins after isoelectric focusing in agarose gels. Electrophoresis 5, 282-285. [6] Kirkeby, S., Moe, D. and Bog-Hansen, T.C. (1993) The silver staining procedure of sodium dodecyl sulfate-gels may be accelerated by shortening fixation time. Electrophoresis 14, 51-55. [7] Heukeshoven, J. and Dernick, R. (1988) Improved silver staining procedure for fast staining in PhastSystem Development Unit I. Staining of dodecyl sulfate gels. Electrophoresis 9, 28-33. [8] Blum, H., Beier, H. and Gross, J.H. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93-99. [9] Heukeshoven, J. and Dernick, R. (1985) Simplified method for silver staining of proteins in polyacrylarnide gels and the mechanism of silver staining. Electrophoresis 6, 103-112. [10] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 227, 680-685. [11] Catty, P. and Deterre, P. (1991) Activation and solubilization of the retinal cGMP-specific phosphodiesterase by limited proteolysis. Eur. J. Bioehem. 199, 263-269. [12] Rabilloud, T. (1990) Mechanisms of protein silver staining in polyacrylamide gels: A 10-year synthesis. Electrophoresis 11. 785-794.
