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Quantitation of proteins by elution of Coomassie brilliant blue R from stained bands after sodium dodecyl sulfate-polyacrylamide gel electrophoresis
ANALYTICAL
BIOCHEMISTRY
1%,23-27
(1986)
Quantitation of Proteins by Elution of Coomassie Brilliant Blue R from Stained Bands after Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis ERICH.BALL Department
qfBiochemistry,
University
of Western
Ontario,
London,
Ontario
N6A 5CI.
Canada
Received July 29. 1985 A simple method for the extraction of Coomassie brilliant blue R from stained protein bands excised from polyactylamide gels is described. Spectrophotometric measurement of the eluted dye forms the basis of a sensitive assayto quantitate proteins in gels in the range 0.5-10 pg. The method requires no unusual equipment and is suitable for measurement of multiple samples. The polypeptide is not extracted and remains available for further analysis. The technique has been applied to three proteins and gels of various acrylamide percentages. 0 1986 Academic Press. Inc. KEY WORDS: sodium dodecyl sulfate-polyacrylamide gels, coomassie brilliant blue, protein determination.
Qualitative examination of the protein composition of complex mixtures by SDS’polyacrylamide gel electrophoresis is a basic analytical tool in protein biochemistry and cellular and molecular biology. After electrophoresis, protein bands are commonly visualized by staining with CBB-R (CI 42660) the most sensitive of several dyes in use (1). It is often desirable to quantitate the amount of protein in particular bands on a gel, and several methods have been developed to accomplish this. Densitometric scanning is perhaps the most common (e.g., 2,3), although several techniques relying on extraction and quantitation of bound dye have been reported (4-8). These quantitation methods suffer from various limitations. The scanning procedure requires access to specialized instrumentation and is subject to errors arising from physical imperfections in the gel as well as distortions of the protein bands (curvature, varying lane widths in slab gels, etc.). Methods of extracting
dyes have utilized pyridine (4), 1 N NaOH at 75°C (5) electroelution (6), or alkaline extraction of naphthol blue black (7,8). Drawbacks to these procedures include the use of noxious solvents, destruction and extraction of protein under basic conditions, or the timeconsuming process of arranging gel slices for electroelution. Common to all these methods as well as the one described here is the problem of variable dye uptake by different proteins. The well-known interference of SDS with staining of polyacrylamide gels suggested that this detergent might be useful in removing CBB-R from protein bands. In this report a simple procedure for the quantitation of protein by extraction of this dye in SDS-isopropanol is described. The method requires no unusual equipment, does not extract the protein, and easily allows the processing of multiple samples. MATERIALS
’ Abbreviations used: SDS, sodium dodecyl sulfate; CBB-R, Coomassie brilliant blue R; TEMED, N,N,l\r,Wtetramethylenediamine; BSA, bovine serum albumin.
Materials.
polyacrylamide 23
AND
METHODS
Chemicals for the preparation of gels were from Bio-Rad Lab0003-2697186 $3.00 Copyright @! 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
24
ERIC
H.
oratories except for ammonium persulfate and TEMED (from Fisher Scientific). CBB-R-250 was also from Bio-Rad. Glycine, Tris, Protein A, and standard proteins (BSA, ovalbumin and &galactosidase) were from Sigma. Na’*‘I was purchased from Amersham and Iodogen from Pierce. The isopropanol used was technical grade from a local supplier. Protein preparation. Standard proteins were weighed, dissolved in distilled water, and diluted with f vol of 3X sample buffer (10% (v/v) glycerol, 5% (v/v) mercaptoethanol, 3% (w/v) SDS, 62 mM Tris-HCl, pH 6.8, and bromphenol blue as a tracking dye). Samples were heated for 3 min in a boiling water bath and serial dilutions in sample buffer were used for electrophoresis. Vinculin was purified to near homogeneity as in Ref. (9). Proteins were iodinated using iodogen (10) and separated from free iodine by molecular sieve chromatography. SDS-polyacrylamide
gel electrophoresis.
The discontinuous system of Laemmli (11) was used in separating gels of 1.5 X 110 X 140 mm with a 3% acrylamide stacking gel of 25 mm. Samples were loaded in wells in the stacking gel formed by a 20-slot comb. Electrophoresis was performed at 35 mA’ constant current until the tracking dye reached the bottom of the separating gel. Staining and destaining. In the standard procedure gels were soaked in about 300 ml (15 gel vol) of a solution of 0.05% CBB-R in 25% (v/v) isopropanol and 10% (v/v) glacial acetic acid for 1 h with gentle agitation on a reciprocal shaker. (This is not enough time for complete penetration of the stain (1) and staining continues during subsequent incubation. Later experiments have shown that total dye uptake is somewhat variable with this short staining time and a longer staining period (2 h for 6 and 10% gels and overnight for 15% gels) is required to consistently attain maximal staining.) The gels were then destained twice by diffusion in 300 ml of 10% methanol and 10% glacial acetic acid (once overnight and once for 8 h) with agitation. The quantity of dye bound to protein after this procedure was
BALL
stable for at least a further 36 h in the destaining solution. Dye elution and measurement. Gel pieces containing a stained protein band (approximate size 5 X 5 mm) were excised with a razor blade and placed in 12 X 75-mm glass test tubes. Some effort was made to keep the slices approximately the same size. One milliliter of an extracting solution (3% SDS in 50% isopropanol in the standard procedure) was added and the tubes capped with Parafilm. They were then placed in an uncovered waterbath set at 37°C for 24 h without agitation. After cooling to room temperature the liquid was removed with a Pasteur pipet and the optical density determined in 1-ml glass cuvettes in a Spectronic 100 1 spectrophotometer using the extracting solution as a blank. Peak absorbance of CBB-R occurred at 595 nm in this solution. Since free dye is assayed metachromatic effects (12) need not be considered. Blank values obtained from similarly sized gel pieces were subtracted from experimental values. In the experiments reported here, blank values ranged from 0.0 15 to 0.034 absorbance units. RESULTS
AND
DISCUSSION
In developing conditions for the extraction of Coomassie brilliant blue from excised protein bands, a variety of parameters were examined. Although SDS alone partially elutes the dye from protein complexes, inclusion of an alcoholic solvent speeds up the process; isopropanol was chosen as being relatively nonirritating. Because other procedures have used alkaline conditions for removing dye (5,7,8), the effects of the pH of the eluting solution were examined. It was found, however, that at a pH greater than 8 the dye suffered a partially irreversible color loss. Others have also noted that high pH decolorizes CBB-R (7). All lower pH values tested (2-8) gave equivalent extraction. Since the gel piece itself was in 10% acetic acid after destaining, no buffer in the eluting solution was needed to keep the pH low. Increasing temperature also
PROTEIN
QUANTITATION
IN POLYACRYLAMIDE
accelerated the removal of the dye from the gel slice; at 37°C the process was twice as fast as at 22°C. The former temperature was chosen for further work since it allowed maximal dye elution in a reasonable time (see below) and was conveniently available. The elution by various concentrations of SDS from BSA in gels of 10 and 15% acrylamide is shown in Fig. 1. The solvent (50% isopropanol) by itself was able to elute some of the dye, but the addition of SDS increased the yield. Increasing SDS concentrations resulted in increasing CBB-R elution up to a maximum at 3%. Control experiments indicated that SDS did not affect the spectral properties of CBB-R in the solvent. The 3% SDS concentration was used in further work. The time course of elution from different percentage acrylamide gels is shown in Fig. 2. The dye was more rapidly removed from the lower percentage gels, as might be expected given their greater porosity. More than half the dye was eluted in all cases within the first hour although a maximal value was not attained until at least 24 h. After 24 h, elution was complete in lower percentage gels and 96% maximal for 15% acrylamide gels. In the interests of convenience and speed, the 24-h time was adopted as standard although slightly submaximal values were attained in 15% gels.
GELS BY DYE ELUTION
25
Time (hrl
FIG. 2. Kinetics of CBB-R elution. Stained gel pieces containing 10 pegof BSA were incubated for various times at 37°C in standard extracting solution. Values are expressed as a percentage of the maximum optical density attained. Slices were cut from 6% (0) 10% (A), and 15% (0) a&amide gels.
It is expected that the use of even higher acrylamide concentrations would require longer elution times. The conditions developed were applied to three proteins to construct standard curves in the range 0- 10 pg protein in 10% acrylamide gels. The results are shown in Fig. 3. All three proteins gave linear plots of absorbance versus
Protean (pg)
FIG. 1. Elution of CBB-R with varying SDS concentrations. Excised gel pieces containing 10 pg of BSA were incubated with the SDS concentrations shown in 50% isopropanol for 9 h at 31°C and the absorbance of the liquid was determined. A, 10%; 0. 15% acrylamide gel.
FIG. 3. Standard curves in 10% acrylamide gels. CBB R was eluted using standard conditions from stained protein bands in 10% acrylamide gels: BSA (A), ovalbumin (O), @-galactosidase(0). Each point represents the average of four slices. Correlation coefficients to the linear regression lines were 0.9995,0.9975, and 0.9996 for BSA, ovalbumin, and @alactosidase, respectively. The average standard deviation of a measurement was 2.9%.
26
ERIC H. BALL
amount of protein, but the slopes of the lines were quite different. This phenomenon of different dye binding by different proteins is well known, and it should be emphasized that a standard curve using the protein of interest should be prepared if absolute (rather than relative) results are desired. The assay will, of course, be more sensitive with proteins which take up more dye. Using the curve generated by varying amounts of BSA for comparative purposes, the present method is more sensitive than other assays relying on dye extraction. One microgram of BSA yields an absorbance of approximately 0.1, compared to 0.0 16 for the naphthol blue black assay (7) and 0.065 for the pyridine extraction assay (4). The procedure described here should have exactly the same sensitivity as the electrophoretic elution of CBB-R (6), because both rely on the same staining reagent, although data for a direct comparison are not available. The sensitivity of this assay is similar to the CBB-R filter assay (13) and somewhat more sensitive than the assay in solution with Coomassie brilliant blue G (14). For a comparison of various other methods, see Table 1 in Ref. (6). The present procedure may be applicable to protein amounts larger than 10 pg (as are other dye elution assays (4-S)); an upper limit for the assay has not been determined. The influence of gel porosity on protein quantitation was investigated using BSA as a standard on gels of 6, 10, and 15% acrylamide (Fig. 4). Among gels subjected to a l-h stain, an identical amount of dye was eluted from BSA in the 6 and 10% gels while less than half as much could be extracted from the protein run on a 15% gel. This was apparently due to the decreased permeability of the high percentage gel to the stain during the limited staining period, since a longer (overnight) stain led to a curve indistinguishable from the 6 and 10% results (Fig. 4). The linear relationship of dye to protein was nevertheless maintained even with the short staining. This suggests that the shape of the curve is not affected by staining time, and a shorter procedure might be utilized at the expense of lower spectropho-
I,0
FIG. 4. Standard curves for BSA in gels of different acrylamide concentration. CBB-R was extracted from gel slices of 6 (X), 10 (0) and 15% (0) acrylamide using the standard procedure. A 15% gel was also subjected to a 15-h staining (A) (rather than the standard 1 h). Each point represents the average from four slices.
tometric readings. For maximum sensitivity a longer staining period should be adopted for acrylamide percentages greater than 10, and probably would also be required for thicker gels. It should also be noted that a certain amount of gel-to-gel variability in the slope of the standard curve (about 10%) has been observed and it is advisable to run a standard together with unknowns on a single gel for quantitative purposes. One source of this inconsistency is the variable amount of free dye remaining during the first destaining stage (depending on the extent of equilibration with the staining solution and the amount of residual stain carried forward to the destaining step), and the variability can be minimized by using longer staining times to ensure complete penetration of the dye (see Materials and Methods). The possibility of coextraction of dye and protein in the isopropanol-SDS solution was examined using several proteins covalently labeled with lz51 as markers (Table 1). In each case, less than 2% of the radioactivity was eluted with the dye. Hence the protein is retained in the gel through the procedure and could be used for further analysis (for example, counting to determine specific activity). The treatment could also be used simply to remove
PROTEIN
QUANTITATION TABLE
IN
POLYACRYLAMIDE
1
‘251-P~~~~~~ EXTRACTION FROM 10% ACRYLAMIDE GELS Radioactivity bW ‘251-Protein
In gel
In liquid
% Retained
Vinculin Ovalbumin Protein A
7608 943
20 4
99.7 99.6
6249
93
98.5
’ Iodinated proteins were mixed with 1 rg of unlabeled protein as carrier and electrophoresed on a 10% gel. After staining and destaining, slices containing the protein were excised and extracted by the standard procedure. The liquid was then removed from the slice and radioactivity was quantitated in the slice and liquid using a gamma counter. Values are the averages from four slices.
Coomassie brilliant blue from proteins, as might be desirable for immunological purposes.
BY
DYE
27
ELUTION
tions involving specific radioactivity determinations of labeled proteins in a mixture. It should also be applicable to protein quantitation in two-dimensional gels where variations in spot shape make scanning difficult. Because the procedure is nondestructive, the polypeptide remaining after dye is removed may also be further analyzed or the procedure may simply be used to remove Coomassie brilliant blue after visualization of a protein’s position in a gel. ACKNOWLEDGMENTS This research was supported by Grant MA 7601 from the Medical Research Council of Canada. The author is a recipient of a Medical Research Council of Canada Scholarship. REFERENCES 1. Wilson, C. M. (1983) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., eds.), Vol. 9 I, Part 1, pp. 236-247, Academic Press, New York. 2. Bennett, J., and Scott, K. J. (1971) Anal. Biochem.
CONCLUSIONS
A very simple method for extracting CBBR from stained protein bands after gel electrophoresis and using the extracted dye for quantitating the protein has been developed. The assay compares favorably with other methods of this type in its simplicity, sensitivity, and ease of use. It is readily adaptable for multiple samples, requires no special equipment or irritating solvents (although isopropanol is not totally innocuous), and relies on the most commonly used protein stain. While the sensitivity of the method varies with different proteins because of the different amounts of dye characteristically bound to them, amounts in the range of 0.5 to 10 fig should be readily determined. The assay is likely to be particularly valuable in applica-
GELS
43, 173-182.
Fishbein, W. N. (1972) Anal. Biochem. 46,388-401. Fenner, C., Traut, R. R., Mason, D. T., and WikmanCoffelt, J. (1975) Anal. Biochem. 63,595-602. 5. Martini, 0. H. W., Kruppa, J., and Temkin, R. (1980) in Electrophoresis ‘79 (Radola, B. J., ed.), pp. 24 I248, de Gruyter, Berlin. 6. Mahoy, J. M., Rieker, J. P., and Rizzo, C. F. (1984) 3. 4.
Anal. Biochem.
141, 503-509.
7.
Everitt, E., and Maksimova, A. (1984) Anal. Biochem.
8.
Racusen. D., and Foote, M. ( 1965) Can&. J. Bat. 43,
141, 17-24. 817-824.
Evans, R. R.. Robson, R. M., and Stromer, M. H. (1984) J. Biol. Chem. 259, 3916-3924. 10. Fraker, P. J.. and Speck, J. C., Jr. (1978) Biochem. 9.
Biophys.
Rex
Commun.
80, 849-857. (London)
11. Laemmh, U. K. (1970) Nature
227, 680-
685.
12. Wilson. C. M. (1979) Anal. Biochem. 96,263-278. 13. McKnight, G. S. (1977) Anal. Biochem. 78, 86-92. 14. Bradford, M. M. (1976) Anal. Biochem. 72,248-254.
BIOCHEMISTRY
1%,23-27
(1986)
Quantitation of Proteins by Elution of Coomassie Brilliant Blue R from Stained Bands after Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis ERICH.BALL Department
qfBiochemistry,
University
of Western
Ontario,
London,
Ontario
N6A 5CI.
Canada
Received July 29. 1985 A simple method for the extraction of Coomassie brilliant blue R from stained protein bands excised from polyactylamide gels is described. Spectrophotometric measurement of the eluted dye forms the basis of a sensitive assayto quantitate proteins in gels in the range 0.5-10 pg. The method requires no unusual equipment and is suitable for measurement of multiple samples. The polypeptide is not extracted and remains available for further analysis. The technique has been applied to three proteins and gels of various acrylamide percentages. 0 1986 Academic Press. Inc. KEY WORDS: sodium dodecyl sulfate-polyacrylamide gels, coomassie brilliant blue, protein determination.
Qualitative examination of the protein composition of complex mixtures by SDS’polyacrylamide gel electrophoresis is a basic analytical tool in protein biochemistry and cellular and molecular biology. After electrophoresis, protein bands are commonly visualized by staining with CBB-R (CI 42660) the most sensitive of several dyes in use (1). It is often desirable to quantitate the amount of protein in particular bands on a gel, and several methods have been developed to accomplish this. Densitometric scanning is perhaps the most common (e.g., 2,3), although several techniques relying on extraction and quantitation of bound dye have been reported (4-8). These quantitation methods suffer from various limitations. The scanning procedure requires access to specialized instrumentation and is subject to errors arising from physical imperfections in the gel as well as distortions of the protein bands (curvature, varying lane widths in slab gels, etc.). Methods of extracting
dyes have utilized pyridine (4), 1 N NaOH at 75°C (5) electroelution (6), or alkaline extraction of naphthol blue black (7,8). Drawbacks to these procedures include the use of noxious solvents, destruction and extraction of protein under basic conditions, or the timeconsuming process of arranging gel slices for electroelution. Common to all these methods as well as the one described here is the problem of variable dye uptake by different proteins. The well-known interference of SDS with staining of polyacrylamide gels suggested that this detergent might be useful in removing CBB-R from protein bands. In this report a simple procedure for the quantitation of protein by extraction of this dye in SDS-isopropanol is described. The method requires no unusual equipment, does not extract the protein, and easily allows the processing of multiple samples. MATERIALS
’ Abbreviations used: SDS, sodium dodecyl sulfate; CBB-R, Coomassie brilliant blue R; TEMED, N,N,l\r,Wtetramethylenediamine; BSA, bovine serum albumin.
Materials.
polyacrylamide 23
AND
METHODS
Chemicals for the preparation of gels were from Bio-Rad Lab0003-2697186 $3.00 Copyright @! 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
24
ERIC
H.
oratories except for ammonium persulfate and TEMED (from Fisher Scientific). CBB-R-250 was also from Bio-Rad. Glycine, Tris, Protein A, and standard proteins (BSA, ovalbumin and &galactosidase) were from Sigma. Na’*‘I was purchased from Amersham and Iodogen from Pierce. The isopropanol used was technical grade from a local supplier. Protein preparation. Standard proteins were weighed, dissolved in distilled water, and diluted with f vol of 3X sample buffer (10% (v/v) glycerol, 5% (v/v) mercaptoethanol, 3% (w/v) SDS, 62 mM Tris-HCl, pH 6.8, and bromphenol blue as a tracking dye). Samples were heated for 3 min in a boiling water bath and serial dilutions in sample buffer were used for electrophoresis. Vinculin was purified to near homogeneity as in Ref. (9). Proteins were iodinated using iodogen (10) and separated from free iodine by molecular sieve chromatography. SDS-polyacrylamide
gel electrophoresis.
The discontinuous system of Laemmli (11) was used in separating gels of 1.5 X 110 X 140 mm with a 3% acrylamide stacking gel of 25 mm. Samples were loaded in wells in the stacking gel formed by a 20-slot comb. Electrophoresis was performed at 35 mA’ constant current until the tracking dye reached the bottom of the separating gel. Staining and destaining. In the standard procedure gels were soaked in about 300 ml (15 gel vol) of a solution of 0.05% CBB-R in 25% (v/v) isopropanol and 10% (v/v) glacial acetic acid for 1 h with gentle agitation on a reciprocal shaker. (This is not enough time for complete penetration of the stain (1) and staining continues during subsequent incubation. Later experiments have shown that total dye uptake is somewhat variable with this short staining time and a longer staining period (2 h for 6 and 10% gels and overnight for 15% gels) is required to consistently attain maximal staining.) The gels were then destained twice by diffusion in 300 ml of 10% methanol and 10% glacial acetic acid (once overnight and once for 8 h) with agitation. The quantity of dye bound to protein after this procedure was
BALL
stable for at least a further 36 h in the destaining solution. Dye elution and measurement. Gel pieces containing a stained protein band (approximate size 5 X 5 mm) were excised with a razor blade and placed in 12 X 75-mm glass test tubes. Some effort was made to keep the slices approximately the same size. One milliliter of an extracting solution (3% SDS in 50% isopropanol in the standard procedure) was added and the tubes capped with Parafilm. They were then placed in an uncovered waterbath set at 37°C for 24 h without agitation. After cooling to room temperature the liquid was removed with a Pasteur pipet and the optical density determined in 1-ml glass cuvettes in a Spectronic 100 1 spectrophotometer using the extracting solution as a blank. Peak absorbance of CBB-R occurred at 595 nm in this solution. Since free dye is assayed metachromatic effects (12) need not be considered. Blank values obtained from similarly sized gel pieces were subtracted from experimental values. In the experiments reported here, blank values ranged from 0.0 15 to 0.034 absorbance units. RESULTS
AND
DISCUSSION
In developing conditions for the extraction of Coomassie brilliant blue from excised protein bands, a variety of parameters were examined. Although SDS alone partially elutes the dye from protein complexes, inclusion of an alcoholic solvent speeds up the process; isopropanol was chosen as being relatively nonirritating. Because other procedures have used alkaline conditions for removing dye (5,7,8), the effects of the pH of the eluting solution were examined. It was found, however, that at a pH greater than 8 the dye suffered a partially irreversible color loss. Others have also noted that high pH decolorizes CBB-R (7). All lower pH values tested (2-8) gave equivalent extraction. Since the gel piece itself was in 10% acetic acid after destaining, no buffer in the eluting solution was needed to keep the pH low. Increasing temperature also
PROTEIN
QUANTITATION
IN POLYACRYLAMIDE
accelerated the removal of the dye from the gel slice; at 37°C the process was twice as fast as at 22°C. The former temperature was chosen for further work since it allowed maximal dye elution in a reasonable time (see below) and was conveniently available. The elution by various concentrations of SDS from BSA in gels of 10 and 15% acrylamide is shown in Fig. 1. The solvent (50% isopropanol) by itself was able to elute some of the dye, but the addition of SDS increased the yield. Increasing SDS concentrations resulted in increasing CBB-R elution up to a maximum at 3%. Control experiments indicated that SDS did not affect the spectral properties of CBB-R in the solvent. The 3% SDS concentration was used in further work. The time course of elution from different percentage acrylamide gels is shown in Fig. 2. The dye was more rapidly removed from the lower percentage gels, as might be expected given their greater porosity. More than half the dye was eluted in all cases within the first hour although a maximal value was not attained until at least 24 h. After 24 h, elution was complete in lower percentage gels and 96% maximal for 15% acrylamide gels. In the interests of convenience and speed, the 24-h time was adopted as standard although slightly submaximal values were attained in 15% gels.
GELS BY DYE ELUTION
25
Time (hrl
FIG. 2. Kinetics of CBB-R elution. Stained gel pieces containing 10 pegof BSA were incubated for various times at 37°C in standard extracting solution. Values are expressed as a percentage of the maximum optical density attained. Slices were cut from 6% (0) 10% (A), and 15% (0) a&amide gels.
It is expected that the use of even higher acrylamide concentrations would require longer elution times. The conditions developed were applied to three proteins to construct standard curves in the range 0- 10 pg protein in 10% acrylamide gels. The results are shown in Fig. 3. All three proteins gave linear plots of absorbance versus
Protean (pg)
FIG. 1. Elution of CBB-R with varying SDS concentrations. Excised gel pieces containing 10 pg of BSA were incubated with the SDS concentrations shown in 50% isopropanol for 9 h at 31°C and the absorbance of the liquid was determined. A, 10%; 0. 15% acrylamide gel.
FIG. 3. Standard curves in 10% acrylamide gels. CBB R was eluted using standard conditions from stained protein bands in 10% acrylamide gels: BSA (A), ovalbumin (O), @-galactosidase(0). Each point represents the average of four slices. Correlation coefficients to the linear regression lines were 0.9995,0.9975, and 0.9996 for BSA, ovalbumin, and @alactosidase, respectively. The average standard deviation of a measurement was 2.9%.
26
ERIC H. BALL
amount of protein, but the slopes of the lines were quite different. This phenomenon of different dye binding by different proteins is well known, and it should be emphasized that a standard curve using the protein of interest should be prepared if absolute (rather than relative) results are desired. The assay will, of course, be more sensitive with proteins which take up more dye. Using the curve generated by varying amounts of BSA for comparative purposes, the present method is more sensitive than other assays relying on dye extraction. One microgram of BSA yields an absorbance of approximately 0.1, compared to 0.0 16 for the naphthol blue black assay (7) and 0.065 for the pyridine extraction assay (4). The procedure described here should have exactly the same sensitivity as the electrophoretic elution of CBB-R (6), because both rely on the same staining reagent, although data for a direct comparison are not available. The sensitivity of this assay is similar to the CBB-R filter assay (13) and somewhat more sensitive than the assay in solution with Coomassie brilliant blue G (14). For a comparison of various other methods, see Table 1 in Ref. (6). The present procedure may be applicable to protein amounts larger than 10 pg (as are other dye elution assays (4-S)); an upper limit for the assay has not been determined. The influence of gel porosity on protein quantitation was investigated using BSA as a standard on gels of 6, 10, and 15% acrylamide (Fig. 4). Among gels subjected to a l-h stain, an identical amount of dye was eluted from BSA in the 6 and 10% gels while less than half as much could be extracted from the protein run on a 15% gel. This was apparently due to the decreased permeability of the high percentage gel to the stain during the limited staining period, since a longer (overnight) stain led to a curve indistinguishable from the 6 and 10% results (Fig. 4). The linear relationship of dye to protein was nevertheless maintained even with the short staining. This suggests that the shape of the curve is not affected by staining time, and a shorter procedure might be utilized at the expense of lower spectropho-
I,0
FIG. 4. Standard curves for BSA in gels of different acrylamide concentration. CBB-R was extracted from gel slices of 6 (X), 10 (0) and 15% (0) acrylamide using the standard procedure. A 15% gel was also subjected to a 15-h staining (A) (rather than the standard 1 h). Each point represents the average from four slices.
tometric readings. For maximum sensitivity a longer staining period should be adopted for acrylamide percentages greater than 10, and probably would also be required for thicker gels. It should also be noted that a certain amount of gel-to-gel variability in the slope of the standard curve (about 10%) has been observed and it is advisable to run a standard together with unknowns on a single gel for quantitative purposes. One source of this inconsistency is the variable amount of free dye remaining during the first destaining stage (depending on the extent of equilibration with the staining solution and the amount of residual stain carried forward to the destaining step), and the variability can be minimized by using longer staining times to ensure complete penetration of the dye (see Materials and Methods). The possibility of coextraction of dye and protein in the isopropanol-SDS solution was examined using several proteins covalently labeled with lz51 as markers (Table 1). In each case, less than 2% of the radioactivity was eluted with the dye. Hence the protein is retained in the gel through the procedure and could be used for further analysis (for example, counting to determine specific activity). The treatment could also be used simply to remove
PROTEIN
QUANTITATION TABLE
IN
POLYACRYLAMIDE
1
‘251-P~~~~~~ EXTRACTION FROM 10% ACRYLAMIDE GELS Radioactivity bW ‘251-Protein
In gel
In liquid
% Retained
Vinculin Ovalbumin Protein A
7608 943
20 4
99.7 99.6
6249
93
98.5
’ Iodinated proteins were mixed with 1 rg of unlabeled protein as carrier and electrophoresed on a 10% gel. After staining and destaining, slices containing the protein were excised and extracted by the standard procedure. The liquid was then removed from the slice and radioactivity was quantitated in the slice and liquid using a gamma counter. Values are the averages from four slices.
Coomassie brilliant blue from proteins, as might be desirable for immunological purposes.
BY
DYE
27
ELUTION
tions involving specific radioactivity determinations of labeled proteins in a mixture. It should also be applicable to protein quantitation in two-dimensional gels where variations in spot shape make scanning difficult. Because the procedure is nondestructive, the polypeptide remaining after dye is removed may also be further analyzed or the procedure may simply be used to remove Coomassie brilliant blue after visualization of a protein’s position in a gel. ACKNOWLEDGMENTS This research was supported by Grant MA 7601 from the Medical Research Council of Canada. The author is a recipient of a Medical Research Council of Canada Scholarship. REFERENCES 1. Wilson, C. M. (1983) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., eds.), Vol. 9 I, Part 1, pp. 236-247, Academic Press, New York. 2. Bennett, J., and Scott, K. J. (1971) Anal. Biochem.
CONCLUSIONS
A very simple method for extracting CBBR from stained protein bands after gel electrophoresis and using the extracted dye for quantitating the protein has been developed. The assay compares favorably with other methods of this type in its simplicity, sensitivity, and ease of use. It is readily adaptable for multiple samples, requires no special equipment or irritating solvents (although isopropanol is not totally innocuous), and relies on the most commonly used protein stain. While the sensitivity of the method varies with different proteins because of the different amounts of dye characteristically bound to them, amounts in the range of 0.5 to 10 fig should be readily determined. The assay is likely to be particularly valuable in applica-
GELS
43, 173-182.
Fishbein, W. N. (1972) Anal. Biochem. 46,388-401. Fenner, C., Traut, R. R., Mason, D. T., and WikmanCoffelt, J. (1975) Anal. Biochem. 63,595-602. 5. Martini, 0. H. W., Kruppa, J., and Temkin, R. (1980) in Electrophoresis ‘79 (Radola, B. J., ed.), pp. 24 I248, de Gruyter, Berlin. 6. Mahoy, J. M., Rieker, J. P., and Rizzo, C. F. (1984) 3. 4.
Anal. Biochem.
141, 503-509.
7.
Everitt, E., and Maksimova, A. (1984) Anal. Biochem.
8.
Racusen. D., and Foote, M. ( 1965) Can&. J. Bat. 43,
141, 17-24. 817-824.
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