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A new staining technique for proteins in polyacrylamide gels using Coomassie brilliant blue G250
ANALYTICAL
BIOCHEMISTRY
82, 580-582
SHORT
(1977)
COMMUNICATIONS
A New Staining Technique for Proteins Gels Using Coomassie Brilliant
in Polyacrylamide Blue G250
A new staining procedure for proteins in polyacrylamide gels has been developed. This procedure, utilizing Coomassie brilliant blue G250, is rapid (as quick as 30 min) and simple to perform, requires little or no destaining, and has a sensitivity of a least 1 pg of protein per band. It can be used to stain proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by polyacrylamide gel electrophoresis in nondissociating solvents. Our staining procedure is based on the procedures developed by Diezel et al. (1) and Malik and Berrie (2). Diezel et al. (1) introduced the use of Coomassie brilliant blue G250, a methyl-substituted triphenylmethane dye. This dye, when used in 12.5% trichloroacetic acid, does not effectively stain polyacrylamide gels, and, thus, the undesirable stained gel backgrounds are not obtained. The interior portions of the protein bands, however, are usually not stained. If, after treatment with this dye, the gels are stored in 5% acetic acid, an intensification of the stained protein bands occurs as the protein-bound dye becomes soluble, diffuses into the gel, and binds again to the interior portions of the protein bands. In our hands, the procedure of Diezel et al. (1) was significantly less sensitive than the conventional procedure using Coomassie brilliant blue R250 (3). Malik and Berrie (2) described a protein-staining procedure with Coomassie brilliant blue R250 prepared using sulfuric acid, KOH, and trichloroacetic acid. With this procedure, rapid staining of the protein bands occurs, but, in our hands, the gel background became significantly stained, and a destaining step with 0.2% (v/v) H&SO, was required to reduce the background. The destaining caused a significant reduction in the intensity of the protein-stained bands. Our staining procedure combines features of both the Diezel et al. (1) and the Malik and Berrie (2) procedures. Stain preparation. Coomassie brilliant blue G250 (xylene brilliant cyanin G) was purchased from K and K Laboratories, Inc. (Catalog No. 27551). To a 0.2% (w/v) aqueous solution of the dye was added an equal volume of 2 N H,SO,. After the solution was mixed well, it was allowed to 580 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN OW3-2697
SHORT
581
COMMUNICATIONS
stand for at least 3 hr. The resulting precipitate was removed by gravity filtration through Whatman No. 1 paper. The volume of the clear brown filtrate was measured and one-ninth volume of 10 N KOH was added, producing a dark purple solution. To the dark purple solution, 100% (w/v) trichloroacetic acid was added to a final concentration of 12% (w/v). The resulting clear light blue solution was ready for use. The staining solution can be stored for several months without loss of effectiveness. It may be reused several times, but the pH of the solution must be maintained below 1.0. Staining procedure. After electrophoresis, the polyacrylamide gel was added directly to the staining solution. For example, a 0.7 x IO-cm cylindrical gel was placed in a test tube with lo-15 ml of stain. Within 30 min, bands containing 5-10 pug of protein were evident. Color development for maximum sensitivity was achieved in 5-8 hr. Gels could be stored in the staining solution without having the gel backgrounds stain
pg PROTEIN
LOADED
ON GEL
FIG. 1. The relationship between the area of the densimetric tracing of the stained protein band and the amount of protein loaded on the polyacrylamide gel. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of denatured bovine serum albumin was performed essentially as described by Weber and Osborn (3). Various amounts of the protein were loaded on cylindrical gels. Electrophoresis was carried out for 4.5 hr at 8 mA/gel. The gels were removed from their electrophoresis tubes, rinsed with water, and then put into test tubes containing 10 ml of Coomassie brilliant blue G250 staining solution. After 5 hr, the gels were removed from the staining solution and were placed in 15 ml of water for 12 hr. Densimetric tracings of the gels at 600 nm were then made using a Gilford linear-transport gel scanner. The areas under the monomeric band of bovine serum albumin, which has a molecular weight of 68,000 and which amounted to about 85-90% of the protein loaded on the gel, were measured. A curve-fitting linear-regression method was used to draw the line in Fig. 1. The coefficient of determination, r*, was 0.99.
582
SHORT COMMUNICATIONS
significantly. If the gels were stored in water (lo-15 ml), there was a marked color intensification of the stained protein bands and gel background reduction producing optimal sensitivity. However, if the gels were placed in solutions containing sulfuric acid, trichloroacetic acid, or acetic acid, there was a rapid loss of intensity of the stained protein bands. Gels containing the protein-stained bands were scanned densimetrically at 600 nm (Fig. 1). A linear relationship was observed between stain intensity and protein concentrations between approximately 1 and 20 pg. If the tracking dye pyronin B was used, the tracking dye was not obscured by the stain, thus permitting direct measurement of relative mobilities. As determined by a direct comparison using bovine serum albumin as the test protein, the sensitivity of the staining procedure reported here using Coomassie brilliant blue G250 was about five times more sensitive than that reported by Malik and Berrie (2). When polyacrylamide gels containing sodium dodecyl sulfate were treated with the staining solution, sodium dodecyl sulfate precipitated in the gels. The resulting opaque background, however, did not obscure the visual detection of most of the stained protein bands. When clear backgrounds were desired, especially for densimetric scanning, the gels were rinsed in several changes of 10% (w/v) trichloroacetic acid-33% (v/v) methanol or in water, prior to treatment with the staining solution. If electrophoresis was performed in solutions that contained not more than 0.1% (v/v) sodium dodecyl sulfate, the amount of opaqueness due to precipitation of the sodium dodecyl sulfate was usually insignificant. ACKNOWLEDGMENTS This work was supported in part by Grant GB-24479A from the National Science Foundation and by National Institutes of Health Training Grant GM-1091. This is Michigan Agricultural Experimental Station Journal Article No. 7727.
REFERliNCES 1. Diezel, W., Kopperschlager. G., and Hoffman, E. (1972) Ana/. Biochern. 2. Malik, N., and Berrie, A. (1972) Anal. Biochem. 49, 173. 3. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406.
48, 617
ROBERT W. BLAKESLEY JOHN A. BOEZI Department of Biochemistry Michigan State University East Lansing, Michigan 48824 Received June 16, 1976; accepted
June
16, 1977.
BIOCHEMISTRY
82, 580-582
SHORT
(1977)
COMMUNICATIONS
A New Staining Technique for Proteins Gels Using Coomassie Brilliant
in Polyacrylamide Blue G250
A new staining procedure for proteins in polyacrylamide gels has been developed. This procedure, utilizing Coomassie brilliant blue G250, is rapid (as quick as 30 min) and simple to perform, requires little or no destaining, and has a sensitivity of a least 1 pg of protein per band. It can be used to stain proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by polyacrylamide gel electrophoresis in nondissociating solvents. Our staining procedure is based on the procedures developed by Diezel et al. (1) and Malik and Berrie (2). Diezel et al. (1) introduced the use of Coomassie brilliant blue G250, a methyl-substituted triphenylmethane dye. This dye, when used in 12.5% trichloroacetic acid, does not effectively stain polyacrylamide gels, and, thus, the undesirable stained gel backgrounds are not obtained. The interior portions of the protein bands, however, are usually not stained. If, after treatment with this dye, the gels are stored in 5% acetic acid, an intensification of the stained protein bands occurs as the protein-bound dye becomes soluble, diffuses into the gel, and binds again to the interior portions of the protein bands. In our hands, the procedure of Diezel et al. (1) was significantly less sensitive than the conventional procedure using Coomassie brilliant blue R250 (3). Malik and Berrie (2) described a protein-staining procedure with Coomassie brilliant blue R250 prepared using sulfuric acid, KOH, and trichloroacetic acid. With this procedure, rapid staining of the protein bands occurs, but, in our hands, the gel background became significantly stained, and a destaining step with 0.2% (v/v) H&SO, was required to reduce the background. The destaining caused a significant reduction in the intensity of the protein-stained bands. Our staining procedure combines features of both the Diezel et al. (1) and the Malik and Berrie (2) procedures. Stain preparation. Coomassie brilliant blue G250 (xylene brilliant cyanin G) was purchased from K and K Laboratories, Inc. (Catalog No. 27551). To a 0.2% (w/v) aqueous solution of the dye was added an equal volume of 2 N H,SO,. After the solution was mixed well, it was allowed to 580 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN OW3-2697
SHORT
581
COMMUNICATIONS
stand for at least 3 hr. The resulting precipitate was removed by gravity filtration through Whatman No. 1 paper. The volume of the clear brown filtrate was measured and one-ninth volume of 10 N KOH was added, producing a dark purple solution. To the dark purple solution, 100% (w/v) trichloroacetic acid was added to a final concentration of 12% (w/v). The resulting clear light blue solution was ready for use. The staining solution can be stored for several months without loss of effectiveness. It may be reused several times, but the pH of the solution must be maintained below 1.0. Staining procedure. After electrophoresis, the polyacrylamide gel was added directly to the staining solution. For example, a 0.7 x IO-cm cylindrical gel was placed in a test tube with lo-15 ml of stain. Within 30 min, bands containing 5-10 pug of protein were evident. Color development for maximum sensitivity was achieved in 5-8 hr. Gels could be stored in the staining solution without having the gel backgrounds stain
pg PROTEIN
LOADED
ON GEL
FIG. 1. The relationship between the area of the densimetric tracing of the stained protein band and the amount of protein loaded on the polyacrylamide gel. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of denatured bovine serum albumin was performed essentially as described by Weber and Osborn (3). Various amounts of the protein were loaded on cylindrical gels. Electrophoresis was carried out for 4.5 hr at 8 mA/gel. The gels were removed from their electrophoresis tubes, rinsed with water, and then put into test tubes containing 10 ml of Coomassie brilliant blue G250 staining solution. After 5 hr, the gels were removed from the staining solution and were placed in 15 ml of water for 12 hr. Densimetric tracings of the gels at 600 nm were then made using a Gilford linear-transport gel scanner. The areas under the monomeric band of bovine serum albumin, which has a molecular weight of 68,000 and which amounted to about 85-90% of the protein loaded on the gel, were measured. A curve-fitting linear-regression method was used to draw the line in Fig. 1. The coefficient of determination, r*, was 0.99.
582
SHORT COMMUNICATIONS
significantly. If the gels were stored in water (lo-15 ml), there was a marked color intensification of the stained protein bands and gel background reduction producing optimal sensitivity. However, if the gels were placed in solutions containing sulfuric acid, trichloroacetic acid, or acetic acid, there was a rapid loss of intensity of the stained protein bands. Gels containing the protein-stained bands were scanned densimetrically at 600 nm (Fig. 1). A linear relationship was observed between stain intensity and protein concentrations between approximately 1 and 20 pg. If the tracking dye pyronin B was used, the tracking dye was not obscured by the stain, thus permitting direct measurement of relative mobilities. As determined by a direct comparison using bovine serum albumin as the test protein, the sensitivity of the staining procedure reported here using Coomassie brilliant blue G250 was about five times more sensitive than that reported by Malik and Berrie (2). When polyacrylamide gels containing sodium dodecyl sulfate were treated with the staining solution, sodium dodecyl sulfate precipitated in the gels. The resulting opaque background, however, did not obscure the visual detection of most of the stained protein bands. When clear backgrounds were desired, especially for densimetric scanning, the gels were rinsed in several changes of 10% (w/v) trichloroacetic acid-33% (v/v) methanol or in water, prior to treatment with the staining solution. If electrophoresis was performed in solutions that contained not more than 0.1% (v/v) sodium dodecyl sulfate, the amount of opaqueness due to precipitation of the sodium dodecyl sulfate was usually insignificant. ACKNOWLEDGMENTS This work was supported in part by Grant GB-24479A from the National Science Foundation and by National Institutes of Health Training Grant GM-1091. This is Michigan Agricultural Experimental Station Journal Article No. 7727.
REFERliNCES 1. Diezel, W., Kopperschlager. G., and Hoffman, E. (1972) Ana/. Biochern. 2. Malik, N., and Berrie, A. (1972) Anal. Biochem. 49, 173. 3. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406.
48, 617
ROBERT W. BLAKESLEY JOHN A. BOEZI Department of Biochemistry Michigan State University East Lansing, Michigan 48824 Received June 16, 1976; accepted
June
16, 1977.