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Quantification of Coomassie Blue stained proteins in polyacrylamide gels based on analyses of eluted dye
ANALYTICAL
BIOCHEMISTRY
63, 595-602
(1975)
Quantification of Coomassie in Polyacrylamide Gels of Eluted
Blue Based
Stained Proteins on Analyses
Dye1
Quantification of Coomassie Brilliant Blue R-250 stained proteins on cellulose acetate strips was made over a limited range of protein concentrations (1); this work extended to quantification of Coomassie Blue stained proteins on polyacrylamide gels (2). Fishbein (3) showed that the major sources of error in quantitative densitometry were protein variations in dye binding, inaccuracy in integrating small areas, and the limited range over which detection of dye was proportional to protein present. Quantification of protein present, based on amino acid composition, was carried out on polyacrylamide gel bands where total nitrogen was based on fluorescamine detection (4). In other studies proteins stained with dibromotrisulphofluorescein (5) allowed for quantification of high protein concentrations, but did not afford a simultaneous comparison of proteins present in low concentrations. With fluorometric scanning of fluorescamine-labeled proteins rapid quantification could be obtained (6), however data again obeyed Beer’s Law only over a limited range of protein concentrations, as observed in other densitometric scanning analyses. This work was undertaken to quantitatively estimate the amount of protein present, based on total nitrogen (7), of specific polypeptides in stained bands of polyacrylamide gels, where there was a large variance in molecular weights of proteins and hence large differences in protein concentrations, as found in the heavy and light subunits of myosin (8-l 1). It was not possible to estimate concentrations of both myosin heavy chains and myosin light chain CZ of canine cardiac left ventricle from densitometric tracings of a single gel; both were not simultaneously within the range of linearity. The restricted range for determining protein concentrations [ 1- 10 Fg (12)] was defined by the limited range over which Coomassie Blue obeyed Beer’s Law. Various errors in densitometric tracings were introduced when proteins were analyzed on separate gels (1,4): varying band widths in a single percent gel introduced large errors. In the procedure presented here dye was extracted from stained bands of polyacrylamide gels with varying volumes of a solvent such as 25% dioxane or 25% pyridine in water (v/v). ’ This work mento-Yolo-Sierra
was supported by Research Heart Association.
Program
Project
59.5 Copyright 0 1975 by Academic Press, Inc. Printed All rights of reproduction in any form reserved.
in the United
States
Grant
HL-14780
and Sacra-
596
SHORT
COMMUNICATIONS
Certain limitations which are present in quantification of proteins in polyacrylamide gels by densitometer tracings are also present with the dye elution technique: (a) As in area analyses of densitometric tracings of overlapping bands (I), there are similar varying degrees of error in dye elution for these types of patterns. (b) Quantification of proteins which have variances in number of dye binding sites ( 1) cannot be overcome unless, as described here, the protein is purified and absorbance relative to protein concentration defined for the purified protein. (c) Nitrogen is determined on the sample applied to the ge1. The assignment of specific activities depends on absorbance ratios between the bands, with neglect of proteins absorbed throughout the gel (13) and of protein retained on the surface. MATERIALS
AND
METHODS
Previously we developed a procedure for purification of canine myocardial myosin by which it was possible to obtained electrophoretically
0l-jl
w-+
0
‘i
; -&-I)---%-
#I -LC*-I)
: -Lc+I)
b
0
c+)
d
e
FIG. 1. Polyacrylamide gel electrophoresis (sodium dodecylsulfate) of purified left ventricular canine cardiac myosin (a); the heavy chains (b); the light chains dissociated from the heavy chains with 8 M urea (c); C, after column chromatography (d): and C2 after column chromatography (e). Techniques for column chromatography were described earlier (15). The myosin chains were analyzed on 6.0% gels as described in earlier studies (18). Protein bands were cut out of each gel as indicated by I.?, and 3 in slot a.
SHORT
597
COMMUNICATIONS
and immunologically pure myosin with high enzymatic activity (14-17). In purification, the tissue was sheared and washed several times in a low salt buffer followed by extraction in 0.3 M KC1 for 15 min; the short extraction period left most of the actin insoluble while still solubilizing the myosin. Myosin was diluted several times to solubilize the troponin and precipitate myosin. Using both sodium pyrophosphate and ATP in the buffer system, myosin, uncomplexed to contaminants, could be specifically precipitated with saturated (NH&SO, adjusted to pH 6.5. Myosin was recovered from the 35-42% (NH&SO, saturation fraction (14-17). Myosin thus purified is shown in Fig. la: light chains were purified using 8 M urea and column chromatography (15). Criteria for myosin purity were described earlier (18). The myosin subunits are shown in Fig. lb-e. For gel electrophoresis a volume of sodium dodecylsulfate buffer (0.1 M Na phosphate buffer, pH 7.2, 1% sodium dodecylsulfate, 1% 2-mercaptoethanol, and 10% glycerol) equivalent to that of the sample was added to either the purified myosin chains, whole myosin, or serum albumin (19). The samples were heated to 85°C for 10 min to dissociate myosin into subunits. The protein subunits were subjected to electrophoresis on 6.0% polyacrylamide, 0.2% N,N’-methylenebisacrylamide in a 0.10 M sodium phosphate buffer, pH 7.2, containing 0.1% sodium dodecylsulfate (19.20). Gels of 6% polyacrylamide (21) were used for quantification of myosin subunits since this percent gel allowed for well-defined bands of both 220,000 (10) and 20,000 (18) molecular weight species simultaneously. Samples containing between 1 and 100 pg of protein were applied to gels in volumes of 50-100 ~1, All procedures for electrophoresis, staining, electrophoretically destaining, and storage of gels were the same as those described by Bickle and Traut (18). Gels were dialyzed in 7.5% acetic acid for 30 min, and stained with Coomassie Brilliant Blue R-250 (1) (made 0.5% Coomassie Blue in 7.5% acetic acid and 50% methanol) for 12-16 hr. For protein quantification by densitometer tracings, gels were scanned at 550 nm in a 10 cm by 1 cm cuvette using the scanning attachment for the Gilford spectrophotometer with a scanning speed of 1 cm/inch and a chart speed of 0.5 min/inch. Elution of dye was performed by cutting out stained bands as shown in Fig. 1, macerating them, extracting the dye with 25% pyridine in water (v/v). The volumes used are given in Fig. 4; it was necessary to bring the solution containing the macerated gel to a defined volume in a graduated tube. Absorption was analyzed between 0.05 and 1.5 absorbance units, the range in which Coomassie Blue obeyed Beer’s Law (Fig. 2)2; pyridine shifted the maximum absorption wavelength (E,,,) from 550 nm (1) to 605 nm. Extraction mixtures were f Solutions were brought for varying gel volumes.
to a defined
volume
in graduated
tubes or flasks
to compensate
598
SHORT COMMUNICATIONS
pg Coamassie
Blue/,l
FIG. 2. Coomassie Brilliant Blue R-250 was dissolved in 25% pyridine. 1% acetic acid, and analyzed for absorbance at varying concentrations to determine the range of linearity in this plot of absorbance vs dye concentration.
shaken overnight at room temperature. Extraction was complete when eluted dye was in equilibrium with that which remained in the macerate gels, as determined by periodic readings3 The eluted dye was analyzed for absorbance at 605 nm and total absorbance was calculated.4 For determination of total nitrogen in the samples applied to the gels, 25 ~1 of the purified protein was hydrolyzed in 0.9 M H,SO, and heated for 45 min at 160°C. Twenty-five microliters of 30% H,O, was added and the mixture was heated to 160°C for 1 hr. Samples were cooled, 200 ~1 of H,O and 100 ,ul of activated Ninhydrin solution (7) were added, heated to 95°C for 20 min, cooled and brought to 10 ml with 50% ethanol. Nitrogen was analyzed at 570 nm against 50% ethanol blanks (7). Assuming 16% nitrogen present in the protein, protein concentrations were calculated. Bovine serum albumin (Sigma Chem. Co.) was used as a standard for comparing myosin subunits relative to binding of dye. RESULTS AND
DISCUSSION
Electrophoresis patterns of purified myosin and each of the purified subunits are shown in Fig. 1. Figure la designates where bands were sliced for dye elution analyses. 3 Time of extraction depended on volume, size of tube or flask used for elution, and degree of shaking. If gels were macerated, band size was not a factor. For all analyses described here elution was complete in 12-16 hr. Absorbance values obtained from background areas were subtracted from all analyses. 4 Total absorbance is defined as: (OD6,,/ml) X (ml/sample).
SHORT
c----/s
50 00 o.24
C. Area Analysis
~~~~
10.0 16 - Light
15 0 pg pmwn 2 4 pg nmogen Chains
c” 020 E 0 LC, A LC2
5 012 “u 008 G 004 ; 016 I///
a.
2
IO 0 16
15.0 )Lg protein 4 t4 “ltroge”
2
50 0.0
$06 % z 05 0 04 E 03 0 z“2 02
a 50 0.0
599
COMMUNICATIONS
Cl Dye Bmding
IO 0 16 Light
15 0 pg proiem 2 4 pg n,trogen Choinr
0 LC, A LCz
01 G(/_
50 00
10.0 16
15.0 pg protein nitrogen 24P’J
FIG. 3. The three proteins, left ventricle myosin heavy chains, myosin light chain C,. and myosin light chain C,, in varying concentrations as indicated, were subjected to electrophoresis on polyacrylamide gels tdodecylsulfate) and stained with Coomassie Blue. This figure shows a correlation between densitometer tracings (A and C) and dye elution (B and D).
Figure 2 shows the range of absorbance units in which Coomassie Brilliant Blue, when dissolved in a solvent such as 25% pyridine, could be quantitated. Concentration of dye was proportional to absorbance up to 1.5 absorbance units. For analysis of both myosin heavy chains and the two myosin light chains, dye binding, as determined by absorbance at 605 nm, was the same for a defined concentration of nitrogen (Fig. 3). When protein concentrations between 1 and 10 pg were analyzed each of the myosin subunits showed similar binding of dye as analyzed either by area analyses of densitometric tracings (Fig. A and C) or dye elution (Fig. B and D). The purified subunits could be used as standards for determining the protein concentration of subunits present in whole myosin. Protein concentrations from 1 to 100 pg could be analyzed by the dye elution method (Fig. 4).5 When determinations were based on area analyses of densitometric tracings, protein concentrations from 1 to 10 Fg of protein could be quantitated (Fig. 4). Using the dye elution procedure one could accurately quantitate the molar ratio of myosin subunits present in left ventricle myosin; such analyses were not possible by integrating the areas of densitometric tracings because of the large variances in molecular weights and variances in protein concentrations between the two 5 Linearity up to 75 pg of protein is shown in Fig. 4, however higher protein concentrations obeyed Beer’s Law; I-100 pg of protein was proportional to absorbance for all proteins analyzed.
600
SHORT COMMUNICATIONS
A Myosin tleovy Chain
50-
I“8 82 fs 4B I-” f5a,,
4.0 -
kg Protein I(Q N Vol (ml)
.75 .60 j -45 30 .5 3 % .I5 IO 1.6 4.0
30 4.8 8.0
50 8.0 14.0
70 I I.2 20.0
90 14.4 25.0
I3 6.01
Bovine Serum Albumin
C 6.0 -
Myosin Light Chain C,
- 75 - 60 - 45
Vdl iml)
40
80
140
200
25.0
FIG. 4. The three proteins, left ventricle myosin heavy chains (A), bovine serum albumin (B), and myosin light chain C, (C) were subjected to electrophoresis on polyacrylamide gels (dodecylsulfate) and stained with Coomassie Blue. Quantification of dye was carried out both by dye elution (a--0-e) and area analyses of the densitometer tracings (A-A-A,.
SHORT
PERCENT PROTEIN ONE-DIMENSIONAL Heavy
a Data
obtained
from
TABLE 1 IN MYOSIN SUBUNITS FROM GEL ELECTROPHORESIS~
chains
90.2 2 0.80 dye
601
COMMUNICATIONS
G
G
5.9 t 0.21
3.9 ? 0.14
solution.
subunits. Table 1 shows the subunit concentrations of left ventricle myosin. The assets of the technique described here include the following: (a) Quantification of protein concentration is independent of the polyacrylamide gel bandwidth (4). [Although there were variances in bandwidths for two proteins differing tenfold or more in molecular weights on a single gel, quantification by dye elution gave accurate results (Table 1); these data were not possible with area analyses of densitometric tracings (4).] (b) The technique presented here increases the range over which protein concentration obeys Beer’s Law on a single gel and thereby limits errors introduced by quantifying two different proteins on two separate gels ( 1,4). (c) With the dye elution technique precise quantification of protein concentrations can be determined from two-dimensional patterns ( 18.23); there is no scanning equipment available for precise quantification of all protein patterns present on two-dimensional gels. REFERENCES 1. FAZEKAS Biophys. 2. CHRAMBACH,
DE ST. GROTH, S., WEBSTER, Acra 71, 377-391.
R. G.,
AND DATYNER,
A. (1966) Anal. Biochem. 15, 544-549. 3. FISHBEIN, W. N. (1972) Anal. Biochem. 46, 388-401. 4. STEIN. S., CHANG, C. H., BOHLER, P., IMAI, S., AND Biochem.
60,
UNDENFRIEND,
A. (1963)
Biochim.
S. (1974)
Anal.
272-277.
5. ORNSTEIN, L. (1964) N. Y. Acad. Sci. 121, 321-349. 6. RAGLAND, W. L., PACE, J. L., AND KEMPER, D. L. (1974) Anal. Biochem. 59, 24-33. 7. SCHIFFMAN, G., KABAT, E. A., AND THOMPSON, W. (1964) Biochemistry 3, I I3- 120. 8. TAYLOR, E. W. (1972) Ann. Rev. Biochem. 41, 577-616. 9. LOWEY, S., AND RISBY, D. (1971) Nature (London) 234, 81-85. 10. DREIZEN, P., AND RICHARDS, D. H. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 29-45. 1 I. SARKAR, S. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 14-17. 12. BICKLE, T. A., AND TRAUT, R. R. (197 I) J. Biol. Chem. 246, 6828-6834. 13. KAPADIA, G., AND CHRAMBACH, A. (1972) Anul. Biochem. 48, 90-102. 14. WIKMAN-COFFELT, J., ZELIS, R., FENNER, C., AND MASON, D. T. (1973) Biochem. Biophys. Res. Commun. 51, 1097-I 104.
602
SHORT COMMUNICATIONS J. ZELIS R., FENNER,
15. WIKMAN-COFFELT, them.
C., AND
MASON,
D. T. (1973)
Prep.
Bio-
3, 439-449.
16. WIKMAN-COFFELT, Chem.
248,
17. FENNER,
J., ZELIS,
C., MASON,
Acad.
Sci.
70,
18. MCPHERSON,
(1974)
R.,
FENNER,
C., AND
MASON,
D. T. (1973) J.
Biol.
5206-5207.
D. T., ZELIS,
J., TRALJT,
J. Biol.
R., AND WIKMAN-COFFELT,
J. (1973)
Proc.
Nat.
3205-3209. Chem.
R.
R., MASON,
249,
D. T., ZELIS,
R., AND WIKMAN-COFFELT,
J.
994-996.
T. A., AND TRAUT, R. R. (1971) .I. Biol. Chem. 246, 6828-6834. 20. WEBER, K., AND OSBORN, M. (1969) J. Bio. Chem. 244,4406-4409. 21. DAVIS, B. J. (1964) N. Y. Acad. Sci. 121,404-427. 22. CHRAMBACH, A., REISFELD, R. A., WYCKOFF, M., AND ZACCARI,
19. BICKLE,
Biochem.
23. HOWARD,
20,
150-
J. (1967)
Anal.
154.
G. A., AND TRAUT,
R. R.
(1973)
FEBS
Lett.
29,
177-180.
CLAUDIA FENNER ROBERT R. TRAUT DEAN T. MASON JOAN WIKMAN-COFFELT Section
of Cardiovascular
Medicine,
Biological
University
qf California,
Medicine, Chemistry, School
Departments and
of
Physiology,
of Medicine,
Davis.
California
Received March 25, 1974; accepted September 6, 1974
BIOCHEMISTRY
63, 595-602
(1975)
Quantification of Coomassie in Polyacrylamide Gels of Eluted
Blue Based
Stained Proteins on Analyses
Dye1
Quantification of Coomassie Brilliant Blue R-250 stained proteins on cellulose acetate strips was made over a limited range of protein concentrations (1); this work extended to quantification of Coomassie Blue stained proteins on polyacrylamide gels (2). Fishbein (3) showed that the major sources of error in quantitative densitometry were protein variations in dye binding, inaccuracy in integrating small areas, and the limited range over which detection of dye was proportional to protein present. Quantification of protein present, based on amino acid composition, was carried out on polyacrylamide gel bands where total nitrogen was based on fluorescamine detection (4). In other studies proteins stained with dibromotrisulphofluorescein (5) allowed for quantification of high protein concentrations, but did not afford a simultaneous comparison of proteins present in low concentrations. With fluorometric scanning of fluorescamine-labeled proteins rapid quantification could be obtained (6), however data again obeyed Beer’s Law only over a limited range of protein concentrations, as observed in other densitometric scanning analyses. This work was undertaken to quantitatively estimate the amount of protein present, based on total nitrogen (7), of specific polypeptides in stained bands of polyacrylamide gels, where there was a large variance in molecular weights of proteins and hence large differences in protein concentrations, as found in the heavy and light subunits of myosin (8-l 1). It was not possible to estimate concentrations of both myosin heavy chains and myosin light chain CZ of canine cardiac left ventricle from densitometric tracings of a single gel; both were not simultaneously within the range of linearity. The restricted range for determining protein concentrations [ 1- 10 Fg (12)] was defined by the limited range over which Coomassie Blue obeyed Beer’s Law. Various errors in densitometric tracings were introduced when proteins were analyzed on separate gels (1,4): varying band widths in a single percent gel introduced large errors. In the procedure presented here dye was extracted from stained bands of polyacrylamide gels with varying volumes of a solvent such as 25% dioxane or 25% pyridine in water (v/v). ’ This work mento-Yolo-Sierra
was supported by Research Heart Association.
Program
Project
59.5 Copyright 0 1975 by Academic Press, Inc. Printed All rights of reproduction in any form reserved.
in the United
States
Grant
HL-14780
and Sacra-
596
SHORT
COMMUNICATIONS
Certain limitations which are present in quantification of proteins in polyacrylamide gels by densitometer tracings are also present with the dye elution technique: (a) As in area analyses of densitometric tracings of overlapping bands (I), there are similar varying degrees of error in dye elution for these types of patterns. (b) Quantification of proteins which have variances in number of dye binding sites ( 1) cannot be overcome unless, as described here, the protein is purified and absorbance relative to protein concentration defined for the purified protein. (c) Nitrogen is determined on the sample applied to the ge1. The assignment of specific activities depends on absorbance ratios between the bands, with neglect of proteins absorbed throughout the gel (13) and of protein retained on the surface. MATERIALS
AND
METHODS
Previously we developed a procedure for purification of canine myocardial myosin by which it was possible to obtained electrophoretically
0l-jl
w-+
0
‘i
; -&-I)---%-
#I -LC*-I)
: -Lc+I)
b
0
c+)
d
e
FIG. 1. Polyacrylamide gel electrophoresis (sodium dodecylsulfate) of purified left ventricular canine cardiac myosin (a); the heavy chains (b); the light chains dissociated from the heavy chains with 8 M urea (c); C, after column chromatography (d): and C2 after column chromatography (e). Techniques for column chromatography were described earlier (15). The myosin chains were analyzed on 6.0% gels as described in earlier studies (18). Protein bands were cut out of each gel as indicated by I.?, and 3 in slot a.
SHORT
597
COMMUNICATIONS
and immunologically pure myosin with high enzymatic activity (14-17). In purification, the tissue was sheared and washed several times in a low salt buffer followed by extraction in 0.3 M KC1 for 15 min; the short extraction period left most of the actin insoluble while still solubilizing the myosin. Myosin was diluted several times to solubilize the troponin and precipitate myosin. Using both sodium pyrophosphate and ATP in the buffer system, myosin, uncomplexed to contaminants, could be specifically precipitated with saturated (NH&SO, adjusted to pH 6.5. Myosin was recovered from the 35-42% (NH&SO, saturation fraction (14-17). Myosin thus purified is shown in Fig. la: light chains were purified using 8 M urea and column chromatography (15). Criteria for myosin purity were described earlier (18). The myosin subunits are shown in Fig. lb-e. For gel electrophoresis a volume of sodium dodecylsulfate buffer (0.1 M Na phosphate buffer, pH 7.2, 1% sodium dodecylsulfate, 1% 2-mercaptoethanol, and 10% glycerol) equivalent to that of the sample was added to either the purified myosin chains, whole myosin, or serum albumin (19). The samples were heated to 85°C for 10 min to dissociate myosin into subunits. The protein subunits were subjected to electrophoresis on 6.0% polyacrylamide, 0.2% N,N’-methylenebisacrylamide in a 0.10 M sodium phosphate buffer, pH 7.2, containing 0.1% sodium dodecylsulfate (19.20). Gels of 6% polyacrylamide (21) were used for quantification of myosin subunits since this percent gel allowed for well-defined bands of both 220,000 (10) and 20,000 (18) molecular weight species simultaneously. Samples containing between 1 and 100 pg of protein were applied to gels in volumes of 50-100 ~1, All procedures for electrophoresis, staining, electrophoretically destaining, and storage of gels were the same as those described by Bickle and Traut (18). Gels were dialyzed in 7.5% acetic acid for 30 min, and stained with Coomassie Brilliant Blue R-250 (1) (made 0.5% Coomassie Blue in 7.5% acetic acid and 50% methanol) for 12-16 hr. For protein quantification by densitometer tracings, gels were scanned at 550 nm in a 10 cm by 1 cm cuvette using the scanning attachment for the Gilford spectrophotometer with a scanning speed of 1 cm/inch and a chart speed of 0.5 min/inch. Elution of dye was performed by cutting out stained bands as shown in Fig. 1, macerating them, extracting the dye with 25% pyridine in water (v/v). The volumes used are given in Fig. 4; it was necessary to bring the solution containing the macerated gel to a defined volume in a graduated tube. Absorption was analyzed between 0.05 and 1.5 absorbance units, the range in which Coomassie Blue obeyed Beer’s Law (Fig. 2)2; pyridine shifted the maximum absorption wavelength (E,,,) from 550 nm (1) to 605 nm. Extraction mixtures were f Solutions were brought for varying gel volumes.
to a defined
volume
in graduated
tubes or flasks
to compensate
598
SHORT COMMUNICATIONS
pg Coamassie
Blue/,l
FIG. 2. Coomassie Brilliant Blue R-250 was dissolved in 25% pyridine. 1% acetic acid, and analyzed for absorbance at varying concentrations to determine the range of linearity in this plot of absorbance vs dye concentration.
shaken overnight at room temperature. Extraction was complete when eluted dye was in equilibrium with that which remained in the macerate gels, as determined by periodic readings3 The eluted dye was analyzed for absorbance at 605 nm and total absorbance was calculated.4 For determination of total nitrogen in the samples applied to the gels, 25 ~1 of the purified protein was hydrolyzed in 0.9 M H,SO, and heated for 45 min at 160°C. Twenty-five microliters of 30% H,O, was added and the mixture was heated to 160°C for 1 hr. Samples were cooled, 200 ~1 of H,O and 100 ,ul of activated Ninhydrin solution (7) were added, heated to 95°C for 20 min, cooled and brought to 10 ml with 50% ethanol. Nitrogen was analyzed at 570 nm against 50% ethanol blanks (7). Assuming 16% nitrogen present in the protein, protein concentrations were calculated. Bovine serum albumin (Sigma Chem. Co.) was used as a standard for comparing myosin subunits relative to binding of dye. RESULTS AND
DISCUSSION
Electrophoresis patterns of purified myosin and each of the purified subunits are shown in Fig. 1. Figure la designates where bands were sliced for dye elution analyses. 3 Time of extraction depended on volume, size of tube or flask used for elution, and degree of shaking. If gels were macerated, band size was not a factor. For all analyses described here elution was complete in 12-16 hr. Absorbance values obtained from background areas were subtracted from all analyses. 4 Total absorbance is defined as: (OD6,,/ml) X (ml/sample).
SHORT
c----/s
50 00 o.24
C. Area Analysis
~~~~
10.0 16 - Light
15 0 pg pmwn 2 4 pg nmogen Chains
c” 020 E 0 LC, A LC2
5 012 “u 008 G 004 ; 016 I///
a.
2
IO 0 16
15.0 )Lg protein 4 t4 “ltroge”
2
50 0.0
$06 % z 05 0 04 E 03 0 z“2 02
a 50 0.0
599
COMMUNICATIONS
Cl Dye Bmding
IO 0 16 Light
15 0 pg proiem 2 4 pg n,trogen Choinr
0 LC, A LCz
01 G(/_
50 00
10.0 16
15.0 pg protein nitrogen 24P’J
FIG. 3. The three proteins, left ventricle myosin heavy chains, myosin light chain C,. and myosin light chain C,, in varying concentrations as indicated, were subjected to electrophoresis on polyacrylamide gels tdodecylsulfate) and stained with Coomassie Blue. This figure shows a correlation between densitometer tracings (A and C) and dye elution (B and D).
Figure 2 shows the range of absorbance units in which Coomassie Brilliant Blue, when dissolved in a solvent such as 25% pyridine, could be quantitated. Concentration of dye was proportional to absorbance up to 1.5 absorbance units. For analysis of both myosin heavy chains and the two myosin light chains, dye binding, as determined by absorbance at 605 nm, was the same for a defined concentration of nitrogen (Fig. 3). When protein concentrations between 1 and 10 pg were analyzed each of the myosin subunits showed similar binding of dye as analyzed either by area analyses of densitometric tracings (Fig. A and C) or dye elution (Fig. B and D). The purified subunits could be used as standards for determining the protein concentration of subunits present in whole myosin. Protein concentrations from 1 to 100 pg could be analyzed by the dye elution method (Fig. 4).5 When determinations were based on area analyses of densitometric tracings, protein concentrations from 1 to 10 Fg of protein could be quantitated (Fig. 4). Using the dye elution procedure one could accurately quantitate the molar ratio of myosin subunits present in left ventricle myosin; such analyses were not possible by integrating the areas of densitometric tracings because of the large variances in molecular weights and variances in protein concentrations between the two 5 Linearity up to 75 pg of protein is shown in Fig. 4, however higher protein concentrations obeyed Beer’s Law; I-100 pg of protein was proportional to absorbance for all proteins analyzed.
600
SHORT COMMUNICATIONS
A Myosin tleovy Chain
50-
I“8 82 fs 4B I-” f5a,,
4.0 -
kg Protein I(Q N Vol (ml)
.75 .60 j -45 30 .5 3 % .I5 IO 1.6 4.0
30 4.8 8.0
50 8.0 14.0
70 I I.2 20.0
90 14.4 25.0
I3 6.01
Bovine Serum Albumin
C 6.0 -
Myosin Light Chain C,
- 75 - 60 - 45
Vdl iml)
40
80
140
200
25.0
FIG. 4. The three proteins, left ventricle myosin heavy chains (A), bovine serum albumin (B), and myosin light chain C, (C) were subjected to electrophoresis on polyacrylamide gels (dodecylsulfate) and stained with Coomassie Blue. Quantification of dye was carried out both by dye elution (a--0-e) and area analyses of the densitometer tracings (A-A-A,.
SHORT
PERCENT PROTEIN ONE-DIMENSIONAL Heavy
a Data
obtained
from
TABLE 1 IN MYOSIN SUBUNITS FROM GEL ELECTROPHORESIS~
chains
90.2 2 0.80 dye
601
COMMUNICATIONS
G
G
5.9 t 0.21
3.9 ? 0.14
solution.
subunits. Table 1 shows the subunit concentrations of left ventricle myosin. The assets of the technique described here include the following: (a) Quantification of protein concentration is independent of the polyacrylamide gel bandwidth (4). [Although there were variances in bandwidths for two proteins differing tenfold or more in molecular weights on a single gel, quantification by dye elution gave accurate results (Table 1); these data were not possible with area analyses of densitometric tracings (4).] (b) The technique presented here increases the range over which protein concentration obeys Beer’s Law on a single gel and thereby limits errors introduced by quantifying two different proteins on two separate gels ( 1,4). (c) With the dye elution technique precise quantification of protein concentrations can be determined from two-dimensional patterns ( 18.23); there is no scanning equipment available for precise quantification of all protein patterns present on two-dimensional gels. REFERENCES 1. FAZEKAS Biophys. 2. CHRAMBACH,
DE ST. GROTH, S., WEBSTER, Acra 71, 377-391.
R. G.,
AND DATYNER,
A. (1966) Anal. Biochem. 15, 544-549. 3. FISHBEIN, W. N. (1972) Anal. Biochem. 46, 388-401. 4. STEIN. S., CHANG, C. H., BOHLER, P., IMAI, S., AND Biochem.
60,
UNDENFRIEND,
A. (1963)
Biochim.
S. (1974)
Anal.
272-277.
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CLAUDIA FENNER ROBERT R. TRAUT DEAN T. MASON JOAN WIKMAN-COFFELT Section
of Cardiovascular
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Biological
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qf California,
Medicine, Chemistry, School
Departments and
of
Physiology,
of Medicine,
Davis.
California
Received March 25, 1974; accepted September 6, 1974