ANALYTICAL
BIOCHEMISTRY
186,285-287
(I%@)
Lowry Protein Assay containing Sodium Dodecyl Sulfate in ~icrotiter Plates for Protein determinations on Fractions from Brain Tissue Charles R. Harrington CambridgeBrain Bunk Laboratory, Department of Psychiatry, University of CambridgeSchoolof Clinical Medicine, Medical ResearchCouncil Centre, Hills Road, CambridgeCB.2ZQH, United Kingdom
Received
December
13,1989
A modified Lowry protein assay which contains sodium dodecyl sulfate (Markwell et al., 1978, Anal. Bio&en. C&7,207-210) has been adapted for use in Q&well microtiter plates. A sp~trophotometer interfaced with a computer is used to plot the standard curve and calculate the protein content of each sample. The method is suitable for measuring 4-40 pg of protein/assay in a volume of 50 ~1. There was no need for prior solubilization of proteins which had been extracted from brain tissue. Test samples, up to 2 1 in triplicate, can be tested on a single microtiter plate plus standards. Alternatively, further samples can be tested separately on extra plates using the standard curve from the original plate for calculations. 0 1990 Academic PRESS, IRC.
Total protein concentration has been determined frequently using the Folin phenol reagent by the procedure originally described by Lowry et al. (1) in 1951. Several modifications of this method have been reported in order to eliminate interferences caused by various detergents and salts in the assay [see review in (2)]. A simpler dyebinding assay described by Bradford (3) suffers from significant protein-to-protein variability (4). Recently, Smith et al. (5) described an alternative method of protein determination in which the Folin phenol reagent is substituted with bicinchoninic acid (BCA),’ providing an assay that is insensitive to the presence of detergents. Several methods have been reported for adapting Lowry (6,7), Bradford (8), and BCA (9) protein assays for use in microtiter plates, which permits the rapid analysis of large numbers of samples. In this study, the adaptation of a modified Lowry protein assay (lo), con’ Abbreviations albumin; SDS, 0~3-2697/90 Copyright All rights
used: BCA, sodium dodecyl
bicinchoninic sulfate.
$3.00 0 1990 by Academic Press, of reproduction in any form
acid,
BSA,
bovine
serum
taining sodium dodecyl sulfate (SDS), to microtiter plates is described. This assay promotes rapid solubilization of proteins during the assay. It has been applied, in this laboratory, to the analysis of protein concentrations in fractions isolated from human brain tissue. Some of these fractions contain sucrose and EDTA at concentrations which will not cause interference. MATERIALS
AND
METHODS
Reagents. Alkaline, SDS-containing copper tartrate reagent (reagent A) (10) was prepared freshly each day by mixing a stock solution of 4% CuSO,- 5Hz0 with 100 vol of a solution containing 2% Na&03, 0.4% NaOH, 0.16% NafK-tartrate, and 1% SDS. Folin-Ciocalteu phenol reagent (BDH, Ltd.) was diluted immediately before use with an equal volume of distilled water. Bovine serum albumin (BSA, fraction V, Sigma A7030) was dissolved in distilled water and its concentration adjusted using an Azsoof 6.6 for 10 mg/ml of BSA. Working dilutions of BSA (2 ml; 0.8 mg/ml) were stored at -20°C. ~~croplate ~w~protei~ assay. Protein solutions in a final volume of 50 ~1 were dispensed in the wells of polystyrene, flat-bottomed 96-well microtiter plates (Corning No. 25860B). Reagent A (150 ~1) was added to all samples using a multichannel dispenser (Titertek) and the mixtures were incubated at room temperature for at least 10 min. In order to avoid bubbles due to detergent, care was taken to add reagent A slowly down the side of the wells and to ensure that all liquid was completely ejected before removing the tips. Diluted phenol reagent (15 ~1) was then added, with rapid mixing. Thorough mixing was achieved by rotation of the dispenser tips in the reaction mixtures while emptying and filling the tips two or three times. The mixtures were then incubated for 45 min at room temperature, in the dark. Just before measuring the absorbance, bubbles were readily 285
Inc. reserved.
286 CURVE EQUATION: A=0.106
FIT:
Quadratic
Cow.
Coeff:
CHARLES
R. HARRINGTON
0.999
CURVE
y = A+B*x+C*xA2 Br0.0326
FIT:
EQUATION: A=1.04
C=-0.000188
log-Log log(y)
Cow.
Coeff:
0.999
= A+B+log(x)
B=O.672
10
A
Log
scale of
00
40 Linear
FIG. 1. or log-log described
scale
of
Computer output of standard curves for determination of BSA protein concentration. (B) coordinates. The absorbance values are the means of triplicate determinations. in the text. The regression coefficients and parameters for the curve fitting equations
removed from the mixtures by directing a stream of air across the surface at an angle of approximately 45”. Standards were included in triplicate from 0 to 40 pg BSA/well in 4-pg increments. This allowed space for 21 unknown samples to be tested, in triplicate, on a single plate. Absorbance at 650 nm was determined using a Vml% kinetic microplate reader (molecular Devices Corp., Palo Alto, CA) and data were analyzed directly using the Softmax program (Molecular Devices). The standard curve was constructed from a plot of the absorbance at 650 nm (AssO)versus the total protein in micrograms per well using a quadratic fit of the curve. Amino-acid analysis of brain tissue proteins. Aminoacid analysis of fractions prepared from brain tissue of patients suffering from Alzheimer’s disease (11,121 was done after hydrolysis, in UUCUO, in 6 M HCl containing 0.1% phenol at 110°C for 16 h using a Durrum Model 600 amino acid analyzer. RESULTS
c
pg
AND
DISCUSSION
A linear scale plot of AGsOversus BSA concentration is shown in Fig. 1A. The plot is nonlinear but a good correlation (regression coefficient = 0.999) was obtained by selecting a quadratic fit for the standard curve. Early versions (1.0) of Softmax did not allow the fitting of data to a linear log-log plot (13) but this is now available on version 2.01 (Fig. 1B). The regression coefficient for both plots is similar. The former method of analysis is still chosen in this laboratory since calculated values above the range of the standard curve (4 to 40 pg protein/well) are not calculated by extrapolation.
The same data are presented using linear (A) Softmax was used to fit curves to the data as are given at the top of each plot.
When the range is extended, the log-log plot is linear from 0.5 to 200 pg/well (regression coefficient = 0.996 for 18 determinants, in triplicate) and the coefficient of variation for values through this range was no greater than 6%. The assay described here has been used to determine protein in fractions from brain tissue which frequently contain low levels of SDS or are not readily soluble. The inclusion of SDS in reagent A (10) allows proteins in such samples to be readily solubilized. The author has also used this assay (10) for measuring protein in bacterial membrane samples without prior solubilization.
TABLE
1
Protein Content of Fractions from Paired Helical Filaments Total
Method” Amino acid analysis Microplate protein assay
protein content(fig)b in fraction:
la
lb
lc
2
105 110
555 600
9 10
445 510
a Protein calculated from total amino acid analyses (cysteine and tryptophan not measured) or by microplate protein assay on the same samples, using BSA as standard. b Pronase-treated paired helical filaments (11) were sonicated in formic acid to yield a soluble, F5.5 fraction (fraction la) and an insoluble fraction that was largely soluble at pH 11.0 (fraction lb) leaving a residual pellet suspended at pH 11.0 (fraction lc). Alternatively, sonication in NH,HCOs yielded short fragments of filaments that were not sedimented at 10,OOOg for 10 min (fraction 2).
PROTEIN
ASSAY IN MICROTITER
An example of the application of this assay to fractions from brain tissue is detailed in Table 1. Insoluble preparations of Pronase-treated paired helical filaments (11) from the brain of a patient who suffered from Alzheimer’s disease were treated by sonication in different solutions: (i) in formic acid a fragment of tau protein termed F5.5 (11,12) is released leaving a residue which is soluble at pH 11.0 and (ii) in 50 mM NHIHCOB (pH 8.0) short fragmented paired helical filaments can be solubilized (12). The protein content of these fractions was analyzed using the microtiter protein assay and by amino acid analysis. There is good agreement for total protein contents estimated by the two methods indicating that proteins were readily solubilized in the protein assay. The modified Lowry method also enables samples containing up to 0.2 M sucrose or 2.5 mM EDTA to be measured without interference (10) which is important for determining the protein in paired helical filaments isolated from sucrose density gradients (11) where sucrose has not been removed completely by dialysis or centrifugation. The critical points for attention in this assay are the thorough and rapid mixing of Folin phenol reagent and the removal of surface bubbles prior to determination of absorbance values. With a computer interface and using Softmax, it is possible to sample very large numbers of protein solutions simultaneously since the standard curve obtained on one microtiter plate can be used to analyze unknown samples on subsequent plates tested at the same time. Multiple serial dilutions of samples
287
PLATES
having unknown protein concentrations can be tested, therefore, with little extra inconvenience. ACKNOWLEDGMENTS The author thanks the Medical Research support and Mrs. Soo West for manuscript
Council, UK, for financial preparation.
REFERENCES 1. Lowry, (1951)
0. J., Rosebrough, N. J., Farr, J. Bid. Chem. 193,265-275.
2. Peterson,
G. L. (1979)
3. Bradford,
M. M. (1976)
4. Keller,
Anal.
Biochem.
Anal.
P. R., and Neville,
A. L., and Randall,
R. J.
100,201-220.
Biochem.
72,248-254.
M. C. (1986)
Clin. Chem.
32,
120-123.
5. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1988) Anal. Biochem. 150,76-85. 6. Fryer, Anal.
H. J. L., Davis, G. E., Manthorpe, Biochem. 153,262-266.
7. Sorensen,
K., and Brodbeck,
M., and Varon,
U. (1986)
8. Redinbaugh, 147,144-147.
M. G., and Campbell,
9. Redinbaugh, 267-271.
M. G., and Turley,
Experientia
W. H. (1985) R. B. (1986)
10. Markwell, M. A. K., Haas, S. M., Bieber, (1978) Anal. Biochem. 87,207-210.
Anal.
S. (1986)
42,161-162. Anal.
Biochem.
Biochem.
L. L., and Tolbert,
153, N. E.
11. Wischik, C. M., Novak, M., Thegersen, H. C., Edwards, P. C., Runswick, M. J., Jakes, R., Walker, J. E., Milstein, C., Roth, M., and Klug, A. (1988) Proc. Natl. Acad. Sci. USA 85,4506-4510. 12. Harrington, preparation. 13. Stauffer,
C. R., Edwards, C. E. (1975)
Anal.
P. C., and Wischik, Biochem.
69,646-649.
C. M.
(1989)
In
BIOCHEMISTRY
186,285-287
(I%@)
Lowry Protein Assay containing Sodium Dodecyl Sulfate in ~icrotiter Plates for Protein determinations on Fractions from Brain Tissue Charles R. Harrington CambridgeBrain Bunk Laboratory, Department of Psychiatry, University of CambridgeSchoolof Clinical Medicine, Medical ResearchCouncil Centre, Hills Road, CambridgeCB.2ZQH, United Kingdom
Received
December
13,1989
A modified Lowry protein assay which contains sodium dodecyl sulfate (Markwell et al., 1978, Anal. Bio&en. C&7,207-210) has been adapted for use in Q&well microtiter plates. A sp~trophotometer interfaced with a computer is used to plot the standard curve and calculate the protein content of each sample. The method is suitable for measuring 4-40 pg of protein/assay in a volume of 50 ~1. There was no need for prior solubilization of proteins which had been extracted from brain tissue. Test samples, up to 2 1 in triplicate, can be tested on a single microtiter plate plus standards. Alternatively, further samples can be tested separately on extra plates using the standard curve from the original plate for calculations. 0 1990 Academic PRESS, IRC.
Total protein concentration has been determined frequently using the Folin phenol reagent by the procedure originally described by Lowry et al. (1) in 1951. Several modifications of this method have been reported in order to eliminate interferences caused by various detergents and salts in the assay [see review in (2)]. A simpler dyebinding assay described by Bradford (3) suffers from significant protein-to-protein variability (4). Recently, Smith et al. (5) described an alternative method of protein determination in which the Folin phenol reagent is substituted with bicinchoninic acid (BCA),’ providing an assay that is insensitive to the presence of detergents. Several methods have been reported for adapting Lowry (6,7), Bradford (8), and BCA (9) protein assays for use in microtiter plates, which permits the rapid analysis of large numbers of samples. In this study, the adaptation of a modified Lowry protein assay (lo), con’ Abbreviations albumin; SDS, 0~3-2697/90 Copyright All rights
used: BCA, sodium dodecyl
bicinchoninic sulfate.
$3.00 0 1990 by Academic Press, of reproduction in any form
acid,
BSA,
bovine
serum
taining sodium dodecyl sulfate (SDS), to microtiter plates is described. This assay promotes rapid solubilization of proteins during the assay. It has been applied, in this laboratory, to the analysis of protein concentrations in fractions isolated from human brain tissue. Some of these fractions contain sucrose and EDTA at concentrations which will not cause interference. MATERIALS
AND
METHODS
Reagents. Alkaline, SDS-containing copper tartrate reagent (reagent A) (10) was prepared freshly each day by mixing a stock solution of 4% CuSO,- 5Hz0 with 100 vol of a solution containing 2% Na&03, 0.4% NaOH, 0.16% NafK-tartrate, and 1% SDS. Folin-Ciocalteu phenol reagent (BDH, Ltd.) was diluted immediately before use with an equal volume of distilled water. Bovine serum albumin (BSA, fraction V, Sigma A7030) was dissolved in distilled water and its concentration adjusted using an Azsoof 6.6 for 10 mg/ml of BSA. Working dilutions of BSA (2 ml; 0.8 mg/ml) were stored at -20°C. ~~croplate ~w~protei~ assay. Protein solutions in a final volume of 50 ~1 were dispensed in the wells of polystyrene, flat-bottomed 96-well microtiter plates (Corning No. 25860B). Reagent A (150 ~1) was added to all samples using a multichannel dispenser (Titertek) and the mixtures were incubated at room temperature for at least 10 min. In order to avoid bubbles due to detergent, care was taken to add reagent A slowly down the side of the wells and to ensure that all liquid was completely ejected before removing the tips. Diluted phenol reagent (15 ~1) was then added, with rapid mixing. Thorough mixing was achieved by rotation of the dispenser tips in the reaction mixtures while emptying and filling the tips two or three times. The mixtures were then incubated for 45 min at room temperature, in the dark. Just before measuring the absorbance, bubbles were readily 285
Inc. reserved.
286 CURVE EQUATION: A=0.106
FIT:
Quadratic
Cow.
Coeff:
CHARLES
R. HARRINGTON
0.999
CURVE
y = A+B*x+C*xA2 Br0.0326
FIT:
EQUATION: A=1.04
C=-0.000188
log-Log log(y)
Cow.
Coeff:
0.999
= A+B+log(x)
B=O.672
10
A
Log
scale of
00
40 Linear
FIG. 1. or log-log described
scale
of
Computer output of standard curves for determination of BSA protein concentration. (B) coordinates. The absorbance values are the means of triplicate determinations. in the text. The regression coefficients and parameters for the curve fitting equations
removed from the mixtures by directing a stream of air across the surface at an angle of approximately 45”. Standards were included in triplicate from 0 to 40 pg BSA/well in 4-pg increments. This allowed space for 21 unknown samples to be tested, in triplicate, on a single plate. Absorbance at 650 nm was determined using a Vml% kinetic microplate reader (molecular Devices Corp., Palo Alto, CA) and data were analyzed directly using the Softmax program (Molecular Devices). The standard curve was constructed from a plot of the absorbance at 650 nm (AssO)versus the total protein in micrograms per well using a quadratic fit of the curve. Amino-acid analysis of brain tissue proteins. Aminoacid analysis of fractions prepared from brain tissue of patients suffering from Alzheimer’s disease (11,121 was done after hydrolysis, in UUCUO, in 6 M HCl containing 0.1% phenol at 110°C for 16 h using a Durrum Model 600 amino acid analyzer. RESULTS
c
pg
AND
DISCUSSION
A linear scale plot of AGsOversus BSA concentration is shown in Fig. 1A. The plot is nonlinear but a good correlation (regression coefficient = 0.999) was obtained by selecting a quadratic fit for the standard curve. Early versions (1.0) of Softmax did not allow the fitting of data to a linear log-log plot (13) but this is now available on version 2.01 (Fig. 1B). The regression coefficient for both plots is similar. The former method of analysis is still chosen in this laboratory since calculated values above the range of the standard curve (4 to 40 pg protein/well) are not calculated by extrapolation.
The same data are presented using linear (A) Softmax was used to fit curves to the data as are given at the top of each plot.
When the range is extended, the log-log plot is linear from 0.5 to 200 pg/well (regression coefficient = 0.996 for 18 determinants, in triplicate) and the coefficient of variation for values through this range was no greater than 6%. The assay described here has been used to determine protein in fractions from brain tissue which frequently contain low levels of SDS or are not readily soluble. The inclusion of SDS in reagent A (10) allows proteins in such samples to be readily solubilized. The author has also used this assay (10) for measuring protein in bacterial membrane samples without prior solubilization.
TABLE
1
Protein Content of Fractions from Paired Helical Filaments Total
Method” Amino acid analysis Microplate protein assay
protein content(fig)b in fraction:
la
lb
lc
2
105 110
555 600
9 10
445 510
a Protein calculated from total amino acid analyses (cysteine and tryptophan not measured) or by microplate protein assay on the same samples, using BSA as standard. b Pronase-treated paired helical filaments (11) were sonicated in formic acid to yield a soluble, F5.5 fraction (fraction la) and an insoluble fraction that was largely soluble at pH 11.0 (fraction lb) leaving a residual pellet suspended at pH 11.0 (fraction lc). Alternatively, sonication in NH,HCOs yielded short fragments of filaments that were not sedimented at 10,OOOg for 10 min (fraction 2).
PROTEIN
ASSAY IN MICROTITER
An example of the application of this assay to fractions from brain tissue is detailed in Table 1. Insoluble preparations of Pronase-treated paired helical filaments (11) from the brain of a patient who suffered from Alzheimer’s disease were treated by sonication in different solutions: (i) in formic acid a fragment of tau protein termed F5.5 (11,12) is released leaving a residue which is soluble at pH 11.0 and (ii) in 50 mM NHIHCOB (pH 8.0) short fragmented paired helical filaments can be solubilized (12). The protein content of these fractions was analyzed using the microtiter protein assay and by amino acid analysis. There is good agreement for total protein contents estimated by the two methods indicating that proteins were readily solubilized in the protein assay. The modified Lowry method also enables samples containing up to 0.2 M sucrose or 2.5 mM EDTA to be measured without interference (10) which is important for determining the protein in paired helical filaments isolated from sucrose density gradients (11) where sucrose has not been removed completely by dialysis or centrifugation. The critical points for attention in this assay are the thorough and rapid mixing of Folin phenol reagent and the removal of surface bubbles prior to determination of absorbance values. With a computer interface and using Softmax, it is possible to sample very large numbers of protein solutions simultaneously since the standard curve obtained on one microtiter plate can be used to analyze unknown samples on subsequent plates tested at the same time. Multiple serial dilutions of samples
287
PLATES
having unknown protein concentrations can be tested, therefore, with little extra inconvenience. ACKNOWLEDGMENTS The author thanks the Medical Research support and Mrs. Soo West for manuscript
Council, UK, for financial preparation.
REFERENCES 1. Lowry, (1951)
0. J., Rosebrough, N. J., Farr, J. Bid. Chem. 193,265-275.
2. Peterson,
G. L. (1979)
3. Bradford,
M. M. (1976)
4. Keller,
Anal.
Biochem.
Anal.
P. R., and Neville,
A. L., and Randall,
R. J.
100,201-220.
Biochem.
72,248-254.
M. C. (1986)
Clin. Chem.
32,
120-123.
5. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1988) Anal. Biochem. 150,76-85. 6. Fryer, Anal.
H. J. L., Davis, G. E., Manthorpe, Biochem. 153,262-266.
7. Sorensen,
K., and Brodbeck,
M., and Varon,
U. (1986)
8. Redinbaugh, 147,144-147.
M. G., and Campbell,
9. Redinbaugh, 267-271.
M. G., and Turley,
Experientia
W. H. (1985) R. B. (1986)
10. Markwell, M. A. K., Haas, S. M., Bieber, (1978) Anal. Biochem. 87,207-210.
Anal.
S. (1986)
42,161-162. Anal.
Biochem.
Biochem.
L. L., and Tolbert,
153, N. E.
11. Wischik, C. M., Novak, M., Thegersen, H. C., Edwards, P. C., Runswick, M. J., Jakes, R., Walker, J. E., Milstein, C., Roth, M., and Klug, A. (1988) Proc. Natl. Acad. Sci. USA 85,4506-4510. 12. Harrington, preparation. 13. Stauffer,
C. R., Edwards, C. E. (1975)
Anal.
P. C., and Wischik, Biochem.
69,646-649.
C. M.
(1989)
In
