- Email: [email protected]
Measurement of protein in cell suspensions using the Commassie brilliant blue dye-binding assay
ANALYTICAL
BIOCHEMISTRY
126, 355-359
(1982)
Measurement of Protein in Cell Suspensions Using the Coomassie Brilliant Blue Dye-Binding Assay GEIR 0. GOGSTAD
AND MAY-BRITT
KRWTNES
Research Institute for Internal Medicine, Section on Hemostasis and Thrombosis, University of Oslo, Rikshospitalet. Oslo, Norway Received March 31, 1982 The direct measurement of protein in cell suspensions using the Coomassie brilliant blue dyebinding assay demonstrated markedly lower values compared to those obtained with the Lowry assay.It is shown that the addition of small amounts of Triton X-100 or NaOH to the cell suspensions prior to addition of the dye reagent corrected this discrepancy. Standards of soluble proteins may be used for the quantitation of protein in cell suspensions with the dyebinding assay provided that the same amounts of Triton X-100 or NaOH are added to both the standards and the samples.
The Coomassie brilliant blue dye-binding assay for the quantitation of protein was introduced by Bradford ( 1). This assay is even more sensitive than the frequently used Lowry assay (2), and a dye-reagent concentrate is commercially available (3). The dyebinding assay is inexpensive, simple to perform, and convenient for routine use because color development occurs in less than 5 min and the color remains stable for at least 1 h. An important advantage of the assay is the lack of interference from many compounds known to interfere with the Lowry assay (34). A summary of these chemicals is given by Peterson (7). Among the few compounds that severely interfere with the dye-binding assay are alkaline buffers, some detergents including sodium dodecyl sulfate and Triton X- 100, and Ampholines (1,3,5). It has been claimed that rather crude samples such as cell or tissue homogenates may be analyzed directly (8,9). Variations in color development between various proteins have been reported (3,6,10,1 l), but this can be minimized by simple modification of the dye reagent ( 12).
355
During analyses of protein in platelet suspensions or suspensions of plateletderived subcellular fractions (13), it was observed that the protein determinations made with the Coomassie brilliant blue dye-binding assay were considerably lower than the corresponding values obtained with the Lowry assay (2). Similar analyses made on samples of soluble proteins showed a good correlation between the two methods, and the dye-binding assay for protein quantitation in cell suspensions therefore appeared to require a renewed evaluation. It was initially observed that the protein determinations became similar when Triton X-100, or NaOH as suggested by Simpson and Sonne (14), was added to the cell suspensions. The inclusion of these substances in the dye-binding assay applied to cell suspensions was therefore further investigated. MATERIALS
AND METHODS
Chemicals. Coomassie brilliant blue reagent concentrate and bovine y-globulin standard were obtained from Bio-Rad Lab-
OOQ3-2697/82/160355-05$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form rcscrvod.
356
GOGSTAD
AND
oratories, Richmond, California. Triton X100 was obtained from Sigma Chemical Company, St. Louis, Missouri. All chemicals were of analytical grade. Platelets. Platelets were prepared from blood collected from healthy, registered blood donors. The blood was anticoagulated with citrate-phosphate-dextrose solution (24.7 mM citric acid/ 142 mrvt disodium citrate/ 25.5 IIIM NaH2P04/204 mM D-glucose) to a final concentration of 12%. Platelet-rich plasma was prepared by centrifugation at mLlax for 15 min. The platelets were sedimented at 25OOg,,,, for 15 min and washed as described elsewhere (13). Finally, the platelets were suspended in 0.154 M NaCl to yield a protein concentration of about 1.5 mg/ml as measured with the Lowry assay (2). Protein preparations. Lyophilized bovine y-globulin standard was solubilized in 0.154 M NaCl to yield a protein concentration of about 1.5 mg/ml as measured with the Lowry assay (2). Human plasma was obtained after removal of platelets from platelet-rich plasma as described in the preceeding paragraph, and the plasma was diluted as for the y-globulin standard to yield a protein concentration of about 1.5 mg/ml as measured with the Lowry assay (2). Dye-binding assay. Bio-Rad dye-reagent concentrate was diluted to 25% with distilled water. Twenty microliters of the protein-containing sample was mixed with 20 ~1 of Triton X-100 or NaOH solutions in varying concentrations at 20°C and incubated for 1 min prior to the addition of 1.0 ml of the diluted dye reagent. The optical density was read at 595 nm in the interval of 5 to 60 min after addition of the dye reagent. Lowry’s protein assay was performed according to the original method (2). RESULTS
AND DISCUSSION
The effect of increasing concentrations of Triton X-100 or NaOH on the net color for-
KRUTNES
mation when a cell suspension was measured with the dye-binding assay was investigated. Human platelets were used as a model system for this investigation. Twenty microliters of Triton X-100 or NaOH solutions were added to 20-~1 samples of platelet suspensions and mixed prior to the addition of 1.0 ml diluted dye reagent. Two platelet concentrations were used. Increasing concentrations of both the solubilizing agents led to increasing color formation (Fig. 1). With Triton X100 the net maximal color was obtained with 20 ~1 of 0.1% solution (final concentration 2 X 10e3%) for both platelet concentrations. With NaOH the net maximal color was obtained with 20~1 of a 0.075 M solution (final concentration 1.5 X 10e3 M), and there was a slight difference in the maximal effect on the two platelet suspensions. The net maximal color obtained with Triton X- 100 was slightly higher than that obtained with NaOH. When a platelet suspension was ultrasonicated and analyzed with the dye-binding assay in the absence of Triton X- 100 or NaOH, the color development was considerably higher than that obtained with control samples of intact platelets, but it never reached the levels obtained in the presence of the solubilizing agents (data not shown). The data thus indicate that the platelets contain proteins which are not available for the dye without prior treatment with cell-lysing, solubilizing agents, or mechanical disruption. It is possible that the dye reagent in the standard procedure reacts primarily with the surface proteins and that the intracellular proteins are more or less unavailable for reaction with the dye. As a consequence, the amounts of protein will be underestimated in this method when platelets are analyzed with soluble protein as standard. When erythrocytes were analyzed, very similar results were obtained, indicating that this conclusion probably can be extended to more cell types. Also, subcellular material, i.e., platelet cY-granules, demonstrated lower
351
PROTEIN ASSAY IN CELL SUSPENSIONS
4 2 TRITON X- 100 (%x 1 OS)
2 0 k 0
0.4
0.2
0.55 x l@mg
PROTEIN
r
NaOtl
(Mx IO?
FIG. 1. The effect of Triton X-100 (upper panel) and NaOH (lower panel) added to platelet suspensions on the color development obtained with the Coomassie briliiant blue dye-binding protein assay. Platelets were suspended in 0.154 M NaCl and analyzed for protein using the Lowry assay. Twenty microliters of the suspensions were mixed with 20 ~1 of Triton X-100 (O0.2%) or NaOH (O-O.1 M) prior to the addition of 1.0 ml dye reagent. The optical density at 595 nm was read against blanks in which 0.154 M NaCl replaced the platelet suspensions. The values are the means of three analyses.
values in the absence than in the presence of solubilizers. However, the difference was less pronounced than with whole cells (data not shown). Such granules, like many other subcellular structures, are vesicles exhibiting surrounding membranes which may prevent intravesicular protein from reaction with the dye. The situation thus resembles that suggested for the reaction of the dye with intact cells. Figure 2 shows that small amounts of Triton X-100 produced a rather high background color, whereas the effect of NaOH was less serious. However, the amounts of solubilizing agents necessary to reach a maximal net color formation did not give a simultaneous rise to severe background color
formation. Thus, the assay might be applicable in the presence of Triton X- 100 as well as NaOH in concentrations sufficient to lyse the cells. To investigate how cell-lysing agents influenced the dye-binding assay, standard curves were prepared using dilution series of either a soluble protein standard (bovine yglobulin fraction), a complex mixture of soluble proteins (human plasma), or a cell suspension (human platelets) in the presence or absence of Triton X-100 or NaOH (Fig. 3). The stock solutions used for the preparation of the dilution series were analyzed for protein with the Lowry assay (2). In the absence of Triton X-100 or NaOH, linear standard curves were obtained, whereas the presence of these compounds resulted in nonlinear standard curves. Only small variations were observed between the curves obtained in the presence of Triton X-100 or NaOH. In the absence of these solubilizing compounds, however, the curve obtained with the platelet suspension was markedly lower than the corresponding curves obtained with either the plasma or the protein standards, as would be
d.5
1:o
1:s
-;a
TRITON X-100 (%x10’) NaOti (Mxlo’)
FIG. 2. The effect of (+) Triton X-100 and (0) NaOH on the background color development with the Coomassie brilliant blue dye-binding assay.Twenty microliters of 0.154 M NaCl and 20-~1 solutions of Triton X-100 (0.05-0.2%) or NaOH (0. l-l .O M) were mixed prior to the addition of 1.0 ml Bio-Rad dye reagent diluted to 25% with distilled water. The optical density was read at 595 nm against a blank in which Triton X-100 or NaOH was replaced by distilled water. The values are the means of three analyses.
358
GOGSTAD
-I
STANDARD
AND KRUTNES PLASMA
l
A
,p” 1 2
1
3
PROTEIN
2
PER
ASSAY
3
(x 10” mgl
FIG. 3. Color development with the Coomassie brilliant blue dye-binding assay applied to a dilution series of soluble protein standard (bovine y-globulin), human plasma, and a platelet suspension. +, Triton X-100 in final concentration 4 X 10e3%; 0, NaOH in final concentration 2 X 10e3 M; m, no addition of solubilizing agent. The protein standard, the plasma, and the platelet suspension were analyzed for protein with the Lowry assay. The stock solutions were diluted with 0.154 M NaCl to obtain dilution series. Twenty microliters of each sample were mixed with 20 pl of either 0.2% Triton X-100 or 0.1 M NaOH or distilled water prior to the addition of 1.O ml diluted dye reagent. The mixtures were read at 595 nm against blanks in which 0. I54 M NaCl replaced the protein samples. The values are the means of three parallels. The experiment was reproduced 3 times using different platelet preparations.
expected from the results shown in Fig. 1. The nonlinearity of the standard curves in the presence of Triton X- 100 and NaOH should be emphasized. With respect to NaOH, this is contradictory to the original work by Bradford (I), who found linear standard curves also in the presence of this compound. However, the commercial reagent used here may be differently composed. This reagent is also reported to interfere with NaOH (3). It was investigated if curves obtained with a soluble protein standard (bovine y-globulin) could be used as standard for complex mixtures of protein or cell suspensions in the presence of Triton X-100 or NaOH. From the data used to construct the curves in Fig. 3 it was readily verified that in the presence of Triton X- 100 or NaOH there exists an apparently linear relationship between the color formed with y-globulin standard, on the one hand, and the color formed with cor-
responding protein concentrations of plasma or platelet suspensions on the other hand. The ratio obtained with the two sets of values were in each case close to 1.O. In the absence of solubilizing agents, a linear relationship and a ratio between the values that was close to i .O were still obtained between the protein standard and plasma, whereas the same ratio was as low as 0.2 1 for the comparison of the protein standard and the platelet suspension. Hence, a standard curve of soluble proteins made in the presence of the same amounts of Triton X- 100 or NaOH as used to solubilize the samples may be used to calculate the protein concentrations of the platelet suspensions. The same conclusion was reached when platelets were replaced by ery-throcytes in the present system, indicating that a similar relationship may exist between soluble protein standards and suspensions of most cell types. Previously, Chiapelli et al. (9) reported lower protein values with crude cell
PROTEIN
ASSAY IN CELL SUSPENSIONS
extracts analyzed with the dye-binding assay compared to corresponding values obtained with the Lowry assay (2). It is possible that this result was due to the same effect as reported in the present study. In conclusion, cell suspensions and probably also most subcellular fractions must be solubilized with a proper agent prior to analyses with the Coomassie brilliant blue dyebinding assay to obtain correct protein quantitations using a soluble protein as standard. In the present study we have shown that Triton X- 100 as well as the previously suggested NaOH (14) may be used for this purpose provided that they are incorporated in the standard solutions as well as in the samples to be measured. ACKNOWLEDGMENTS This work was supported by The Norwegian Council on Cardiovascular Diseases and Anders Jahres Fond til Vitenskapens Fremme.
REFERENCES 1. Bradford, M. M. (1976) Anal. Biochem. 72, 248254.
2.
359
Lowry, 0. H., Rosebrough, N. J., Fat-r, A. L., and Randall, R. J. (195 1) J. Biol. Chem. 193, 265275.
Bio-Rad protein assay (1979) Bio-Rad Technical Bulletin No. 1069 EG, Bio-Rad Laboratories, Richmond, California. 4. Gogstad, G. 0. (1980) Anal. Biochem. 106, 5243.
528.
Spector, T. (1978) Anal. Biochem. 86, 142-146. 6. Sedmak, J. J., and Grossberg, S. E. (1977) Anal. Biochem. 79, 544-552. 7. Peterson, G. L. (1979) Anal. Biochem. 100, 2015.
220.
Pollard, H. B., Menard, R., Brandt, H. A., Pazoles, C. J., Creutz, C. J., and Ramu, A. (1978) Anal. Biochem. 86,761-763. 9. Chiapelli, F., Vasil, A., and Haggerty, D. F. (1979) Anal. B&hem. 94, 160-165. 10. Pierce, J., and Suelter, C. H. (1977) Anal. Biochem. 8.
81,478-480.
11. van Kley, H., and Hale, S. M. ( 1977) Anal. B&hem. t&485-487. Read, S. M., and Northcote, D. H. (198 1) Anal. Biochem. 116, 53-64. 13. Gogstad, G. 0. (1981) Thromb. Res. 20,669-681. 14. Simpson, I. A., and Sonne, 0. (1982) Anal. Biochem. 12.
119,424-427.
BIOCHEMISTRY
126, 355-359
(1982)
Measurement of Protein in Cell Suspensions Using the Coomassie Brilliant Blue Dye-Binding Assay GEIR 0. GOGSTAD
AND MAY-BRITT
KRWTNES
Research Institute for Internal Medicine, Section on Hemostasis and Thrombosis, University of Oslo, Rikshospitalet. Oslo, Norway Received March 31, 1982 The direct measurement of protein in cell suspensions using the Coomassie brilliant blue dyebinding assay demonstrated markedly lower values compared to those obtained with the Lowry assay.It is shown that the addition of small amounts of Triton X-100 or NaOH to the cell suspensions prior to addition of the dye reagent corrected this discrepancy. Standards of soluble proteins may be used for the quantitation of protein in cell suspensions with the dyebinding assay provided that the same amounts of Triton X-100 or NaOH are added to both the standards and the samples.
The Coomassie brilliant blue dye-binding assay for the quantitation of protein was introduced by Bradford ( 1). This assay is even more sensitive than the frequently used Lowry assay (2), and a dye-reagent concentrate is commercially available (3). The dyebinding assay is inexpensive, simple to perform, and convenient for routine use because color development occurs in less than 5 min and the color remains stable for at least 1 h. An important advantage of the assay is the lack of interference from many compounds known to interfere with the Lowry assay (34). A summary of these chemicals is given by Peterson (7). Among the few compounds that severely interfere with the dye-binding assay are alkaline buffers, some detergents including sodium dodecyl sulfate and Triton X- 100, and Ampholines (1,3,5). It has been claimed that rather crude samples such as cell or tissue homogenates may be analyzed directly (8,9). Variations in color development between various proteins have been reported (3,6,10,1 l), but this can be minimized by simple modification of the dye reagent ( 12).
355
During analyses of protein in platelet suspensions or suspensions of plateletderived subcellular fractions (13), it was observed that the protein determinations made with the Coomassie brilliant blue dye-binding assay were considerably lower than the corresponding values obtained with the Lowry assay (2). Similar analyses made on samples of soluble proteins showed a good correlation between the two methods, and the dye-binding assay for protein quantitation in cell suspensions therefore appeared to require a renewed evaluation. It was initially observed that the protein determinations became similar when Triton X-100, or NaOH as suggested by Simpson and Sonne (14), was added to the cell suspensions. The inclusion of these substances in the dye-binding assay applied to cell suspensions was therefore further investigated. MATERIALS
AND METHODS
Chemicals. Coomassie brilliant blue reagent concentrate and bovine y-globulin standard were obtained from Bio-Rad Lab-
OOQ3-2697/82/160355-05$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form rcscrvod.
356
GOGSTAD
AND
oratories, Richmond, California. Triton X100 was obtained from Sigma Chemical Company, St. Louis, Missouri. All chemicals were of analytical grade. Platelets. Platelets were prepared from blood collected from healthy, registered blood donors. The blood was anticoagulated with citrate-phosphate-dextrose solution (24.7 mM citric acid/ 142 mrvt disodium citrate/ 25.5 IIIM NaH2P04/204 mM D-glucose) to a final concentration of 12%. Platelet-rich plasma was prepared by centrifugation at mLlax for 15 min. The platelets were sedimented at 25OOg,,,, for 15 min and washed as described elsewhere (13). Finally, the platelets were suspended in 0.154 M NaCl to yield a protein concentration of about 1.5 mg/ml as measured with the Lowry assay (2). Protein preparations. Lyophilized bovine y-globulin standard was solubilized in 0.154 M NaCl to yield a protein concentration of about 1.5 mg/ml as measured with the Lowry assay (2). Human plasma was obtained after removal of platelets from platelet-rich plasma as described in the preceeding paragraph, and the plasma was diluted as for the y-globulin standard to yield a protein concentration of about 1.5 mg/ml as measured with the Lowry assay (2). Dye-binding assay. Bio-Rad dye-reagent concentrate was diluted to 25% with distilled water. Twenty microliters of the protein-containing sample was mixed with 20 ~1 of Triton X-100 or NaOH solutions in varying concentrations at 20°C and incubated for 1 min prior to the addition of 1.0 ml of the diluted dye reagent. The optical density was read at 595 nm in the interval of 5 to 60 min after addition of the dye reagent. Lowry’s protein assay was performed according to the original method (2). RESULTS
AND DISCUSSION
The effect of increasing concentrations of Triton X-100 or NaOH on the net color for-
KRUTNES
mation when a cell suspension was measured with the dye-binding assay was investigated. Human platelets were used as a model system for this investigation. Twenty microliters of Triton X-100 or NaOH solutions were added to 20-~1 samples of platelet suspensions and mixed prior to the addition of 1.0 ml diluted dye reagent. Two platelet concentrations were used. Increasing concentrations of both the solubilizing agents led to increasing color formation (Fig. 1). With Triton X100 the net maximal color was obtained with 20 ~1 of 0.1% solution (final concentration 2 X 10e3%) for both platelet concentrations. With NaOH the net maximal color was obtained with 20~1 of a 0.075 M solution (final concentration 1.5 X 10e3 M), and there was a slight difference in the maximal effect on the two platelet suspensions. The net maximal color obtained with Triton X- 100 was slightly higher than that obtained with NaOH. When a platelet suspension was ultrasonicated and analyzed with the dye-binding assay in the absence of Triton X- 100 or NaOH, the color development was considerably higher than that obtained with control samples of intact platelets, but it never reached the levels obtained in the presence of the solubilizing agents (data not shown). The data thus indicate that the platelets contain proteins which are not available for the dye without prior treatment with cell-lysing, solubilizing agents, or mechanical disruption. It is possible that the dye reagent in the standard procedure reacts primarily with the surface proteins and that the intracellular proteins are more or less unavailable for reaction with the dye. As a consequence, the amounts of protein will be underestimated in this method when platelets are analyzed with soluble protein as standard. When erythrocytes were analyzed, very similar results were obtained, indicating that this conclusion probably can be extended to more cell types. Also, subcellular material, i.e., platelet cY-granules, demonstrated lower
351
PROTEIN ASSAY IN CELL SUSPENSIONS
4 2 TRITON X- 100 (%x 1 OS)
2 0 k 0
0.4
0.2
0.55 x l@mg
PROTEIN
r
NaOtl
(Mx IO?
FIG. 1. The effect of Triton X-100 (upper panel) and NaOH (lower panel) added to platelet suspensions on the color development obtained with the Coomassie briliiant blue dye-binding protein assay. Platelets were suspended in 0.154 M NaCl and analyzed for protein using the Lowry assay. Twenty microliters of the suspensions were mixed with 20 ~1 of Triton X-100 (O0.2%) or NaOH (O-O.1 M) prior to the addition of 1.0 ml dye reagent. The optical density at 595 nm was read against blanks in which 0.154 M NaCl replaced the platelet suspensions. The values are the means of three analyses.
values in the absence than in the presence of solubilizers. However, the difference was less pronounced than with whole cells (data not shown). Such granules, like many other subcellular structures, are vesicles exhibiting surrounding membranes which may prevent intravesicular protein from reaction with the dye. The situation thus resembles that suggested for the reaction of the dye with intact cells. Figure 2 shows that small amounts of Triton X-100 produced a rather high background color, whereas the effect of NaOH was less serious. However, the amounts of solubilizing agents necessary to reach a maximal net color formation did not give a simultaneous rise to severe background color
formation. Thus, the assay might be applicable in the presence of Triton X- 100 as well as NaOH in concentrations sufficient to lyse the cells. To investigate how cell-lysing agents influenced the dye-binding assay, standard curves were prepared using dilution series of either a soluble protein standard (bovine yglobulin fraction), a complex mixture of soluble proteins (human plasma), or a cell suspension (human platelets) in the presence or absence of Triton X-100 or NaOH (Fig. 3). The stock solutions used for the preparation of the dilution series were analyzed for protein with the Lowry assay (2). In the absence of Triton X-100 or NaOH, linear standard curves were obtained, whereas the presence of these compounds resulted in nonlinear standard curves. Only small variations were observed between the curves obtained in the presence of Triton X-100 or NaOH. In the absence of these solubilizing compounds, however, the curve obtained with the platelet suspension was markedly lower than the corresponding curves obtained with either the plasma or the protein standards, as would be
d.5
1:o
1:s
-;a
TRITON X-100 (%x10’) NaOti (Mxlo’)
FIG. 2. The effect of (+) Triton X-100 and (0) NaOH on the background color development with the Coomassie brilliant blue dye-binding assay.Twenty microliters of 0.154 M NaCl and 20-~1 solutions of Triton X-100 (0.05-0.2%) or NaOH (0. l-l .O M) were mixed prior to the addition of 1.0 ml Bio-Rad dye reagent diluted to 25% with distilled water. The optical density was read at 595 nm against a blank in which Triton X-100 or NaOH was replaced by distilled water. The values are the means of three analyses.
358
GOGSTAD
-I
STANDARD
AND KRUTNES PLASMA
l
A
,p” 1 2
1
3
PROTEIN
2
PER
ASSAY
3
(x 10” mgl
FIG. 3. Color development with the Coomassie brilliant blue dye-binding assay applied to a dilution series of soluble protein standard (bovine y-globulin), human plasma, and a platelet suspension. +, Triton X-100 in final concentration 4 X 10e3%; 0, NaOH in final concentration 2 X 10e3 M; m, no addition of solubilizing agent. The protein standard, the plasma, and the platelet suspension were analyzed for protein with the Lowry assay. The stock solutions were diluted with 0.154 M NaCl to obtain dilution series. Twenty microliters of each sample were mixed with 20 pl of either 0.2% Triton X-100 or 0.1 M NaOH or distilled water prior to the addition of 1.O ml diluted dye reagent. The mixtures were read at 595 nm against blanks in which 0. I54 M NaCl replaced the protein samples. The values are the means of three parallels. The experiment was reproduced 3 times using different platelet preparations.
expected from the results shown in Fig. 1. The nonlinearity of the standard curves in the presence of Triton X- 100 and NaOH should be emphasized. With respect to NaOH, this is contradictory to the original work by Bradford (I), who found linear standard curves also in the presence of this compound. However, the commercial reagent used here may be differently composed. This reagent is also reported to interfere with NaOH (3). It was investigated if curves obtained with a soluble protein standard (bovine y-globulin) could be used as standard for complex mixtures of protein or cell suspensions in the presence of Triton X-100 or NaOH. From the data used to construct the curves in Fig. 3 it was readily verified that in the presence of Triton X- 100 or NaOH there exists an apparently linear relationship between the color formed with y-globulin standard, on the one hand, and the color formed with cor-
responding protein concentrations of plasma or platelet suspensions on the other hand. The ratio obtained with the two sets of values were in each case close to 1.O. In the absence of solubilizing agents, a linear relationship and a ratio between the values that was close to i .O were still obtained between the protein standard and plasma, whereas the same ratio was as low as 0.2 1 for the comparison of the protein standard and the platelet suspension. Hence, a standard curve of soluble proteins made in the presence of the same amounts of Triton X- 100 or NaOH as used to solubilize the samples may be used to calculate the protein concentrations of the platelet suspensions. The same conclusion was reached when platelets were replaced by ery-throcytes in the present system, indicating that a similar relationship may exist between soluble protein standards and suspensions of most cell types. Previously, Chiapelli et al. (9) reported lower protein values with crude cell
PROTEIN
ASSAY IN CELL SUSPENSIONS
extracts analyzed with the dye-binding assay compared to corresponding values obtained with the Lowry assay (2). It is possible that this result was due to the same effect as reported in the present study. In conclusion, cell suspensions and probably also most subcellular fractions must be solubilized with a proper agent prior to analyses with the Coomassie brilliant blue dyebinding assay to obtain correct protein quantitations using a soluble protein as standard. In the present study we have shown that Triton X- 100 as well as the previously suggested NaOH (14) may be used for this purpose provided that they are incorporated in the standard solutions as well as in the samples to be measured. ACKNOWLEDGMENTS This work was supported by The Norwegian Council on Cardiovascular Diseases and Anders Jahres Fond til Vitenskapens Fremme.
REFERENCES 1. Bradford, M. M. (1976) Anal. Biochem. 72, 248254.
2.
359
Lowry, 0. H., Rosebrough, N. J., Fat-r, A. L., and Randall, R. J. (195 1) J. Biol. Chem. 193, 265275.
Bio-Rad protein assay (1979) Bio-Rad Technical Bulletin No. 1069 EG, Bio-Rad Laboratories, Richmond, California. 4. Gogstad, G. 0. (1980) Anal. Biochem. 106, 5243.
528.
Spector, T. (1978) Anal. Biochem. 86, 142-146. 6. Sedmak, J. J., and Grossberg, S. E. (1977) Anal. Biochem. 79, 544-552. 7. Peterson, G. L. (1979) Anal. Biochem. 100, 2015.
220.
Pollard, H. B., Menard, R., Brandt, H. A., Pazoles, C. J., Creutz, C. J., and Ramu, A. (1978) Anal. Biochem. 86,761-763. 9. Chiapelli, F., Vasil, A., and Haggerty, D. F. (1979) Anal. B&hem. 94, 160-165. 10. Pierce, J., and Suelter, C. H. (1977) Anal. Biochem. 8.
81,478-480.
11. van Kley, H., and Hale, S. M. ( 1977) Anal. B&hem. t&485-487. Read, S. M., and Northcote, D. H. (198 1) Anal. Biochem. 116, 53-64. 13. Gogstad, G. 0. (1981) Thromb. Res. 20,669-681. 14. Simpson, I. A., and Sonne, 0. (1982) Anal. Biochem. 12.
119,424-427.