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Quantitative densitometry of 1–50 μg protein in acrylamide gel slabs with coomassie blue
ANALYTICAL
46,
BIOCHEMISTRY
Quantitative
388401
(1972)
Acrylamide
Gel
Slabs
WILLIAM Biochemistry
Branch,
of l-50
Densitometry
Armed
Forces
with
rug Protein
Coomassie
in
Blue’
N. FISHBEIN Institute
Received
April
of
Pathology,
Washington,
D. C. .80306
5, 1971
Polyacrylamide gel electrophoresis is the most sensit’ive technique presently available for separating proteins, and Coomassie blue is the most sensitive protein stain available (1). Together they are regularly used to separate and identify microgram quantities of proteins. Unfortunately, Coomassie blue has been reported to follow Beer’s law over only a narrow concentration range (1,2), and other less sensitive dyes have been suggest,ed for quantitation over a wider range (3). However, this involves duplicating all runs, since the great sensitivity of Coomassie blue is not readily forsaken, or else working at lo-fold greater protein concentrations, if feasible. It would obviously be an advantage to use Coomassie blue both for identification and for quantitation over a reasonable range, and this has impelled the present study. Of more general concern is the lack of convincing evidence as to the accuracy of quantitative dye densitometry, despite the widespread use of this procedure. The major sources of error in quantitative densitometry may be summarized as follows: (a) variation in the amount of dye bound from one protein to another, (b) baseline selection and peak-splitting procedures whenever there is no baseline segment between peaks, (c) nonlinearity in the detector and/or pen response to absorbance, (d) integrator inaccuracy, especially for small areas. The first problem is absent if one deals with a family of isologous proteins, i.e., a single protein which assumes multiple forms. The most readily diagnosed case of such a family is an enzyme displaying non’ This work was supported in part by grant AM-10900 from the United States Public Health Service and in part by a research contract, Project 3AO61102B71P-06, from the United States Army Medical Research and Development Command. The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. 388 @ 1972 by Academic
Press.
Inc.
QUANTITATIVE
DENSITOMEX’RY
OF
PROTEIN
389
genetic isozymes, where the same primary structure in all forms would dictate equivalent dye-binding for all bands. Thus the quantitation of a series of enzyme polymers should offer a most favorable situation for densitometry. Of specific interest here is the enzyme urease, which can be prepared in a form homogeneous by both electrophoresis and ultracentrifugation (4), and from which can be produced a polymer series, as well as other nongenetic isozymes (5,6). Since the bands are well separated on electrophoresis, there is no difficulty in baseline estimation and no need for peak splitting; so t’he second source of error is also absent. The third and fourth objections were removed by standardizing the densit’ometer with a series of neutral density filters, and by the use of repetitive digital integration. Using therefore the most favorable possible conditions for densitometric accuracy, we can test whether the dye uptake is proportional to the amount of protein applied, and more rigorously, we can test the most obvious question to which we have seen no answer in the literature: is the dye uptake of a protein independent of its migration distance, and hence of its band width? As will be seen below, the answer to this latter question is a depressing “no” in the case of Coomassie blue, although ureas@ can nevertheless be quantitated after suitable corrections. MATERIALS
AND
METHODS
Chemicals Pure ol-urease, the monomer with molecular weight 480,000, was prepared from jackbean meal as previously described (4) and the protein content was determined with the Folin-phenol reagent (7). Coomassieblue was obtained from Mann Research Laboratories (New York, N. Y.) and prepared as 1% stock solutions in absolute ethanol. Thyroglobulin was also obtained from Mann Research Laboratories; and 3X crystallized bovine serum albumin was obtained from Pentex (Kankakee, Ill.). Stock solutions of 60% trichloroacetic acid were prepared in water and diluted 1:5 as needed. Electrophoresis Vertical .gel slab electrophoresis assemblies (EC Apparatus Corp., Philadelphia, Pa.) were cooled by circulating refrigerated water to maintain a gel temperature of 10” throughout the run, A mixture of 95% acrylamide/5% methylene bisacrylamide was used in all experiments, and was weighed in an analytical balance to obtain 5% or 6% acrylamide gels, the former being used for all standard quantitations. Ammonium
390
WILLIAM
N.
FISHBEIN
persulfate and N,N,N’,N’-tetramethylethylenediamine were used to catalyze polymerization (6)) and all gels were prerun for at least 1 hr at 300 V before sample application. The same buffer was used for gel preparation and in the tank reservoirs: 0.05 M Tris-acetate, pH 8.5, with 1 miU EDTA ; buffer reservoirs were continuously pump-exchanged throughout all runs. Protein solutions were weighted by addition of an equal volume of 50% sucrose in water, and were applied with Hamilton syringes, keeping the needle tip at the extreme upper lip of each slot opening. With the electrophoresis assembly illuminated from behind, the refraction lines of the sucrose solution were readily observed, and volumes of l-60 ~1 could be applied in a slot with high accuracy. In many experiments, after a l-2 hr electrophoresis, the voltage and buffer pumps were turned off, a second series of samples was applied, and further electrophoresis was then carried out. This could be repeated once or twice more, depending on the electrophoresis intervals selected. Staining
and Photography
The gels were fixed and stained by gentle rocking overnight in plastic boxes containing a solution prepared by diluting 1% Coomassie blue in absolute ethanol 20-fold with 12% trichloroacetic acid. Protein staining was not significantly increased on prolonging the staining period beyond 12 hr (up to 72 hr), but 4-6 hr was definitely insufficient time for full staining. After rinsing out the dye with distilled water, the gel was removed a;ld the tray was rinsed several times with methanol and then water before replacing the gel for overnight destaining in 12% trichloroacetic acid. A second overnight destaining was normally sufficient to completely clear the gel background. After destaining, the gels were equilibrated with water, and photographed with an MP-3 Polaroid system, using a pink-tinted transilluminator (Cole-Parmer, Chicago, Ill.) ; no filter was necessary. Other methods of staining and destaining with Coomassie blue were found wanting when visually compared to that above, using duplicate samples and a split-gel technique. Electrophoretic destaining was variable and uneven. The use of aqueous Coomassie blue after prior gel fixing (8) was definitely less sensitive, even when much higher concentrations of the dye were used. Hydrochloric acid (1 N) was not effective in destaining the gel background, although free dye was more soluble in this acid than in trichloroacetic acid. On the other hand, destaining by any method was extremely difficult after overnight storage in 6% aqueous acetic acid. Aqueous stock solutions of Coomassie blue (2) gradually decomposed to yield a gummy residue; the reaction was accelerated in trichloroacetic acid. and retarded in ethanol.
QUANTITATIVE
DENSITOMETRY
Densitometry
OF
PROTEIN
391
and Integration
A Photovolt model 542 Densicord was equipped with a fine resolution slit (0.1 X 6 mm), starch gel carriage geared to 0.5 in./min travel, chart expansion accessory set at 2.411 expansion, and a 570 nm filter, which gave maximum absorption for Coomassie blue stained proteins. The Photovolt disc integrat,or was found to be erratic and unreliable, and the chart strips were instead integrated with the Technicon model AAG digital integrator in the linear mode, with the chart speed set at 4.5 in./ min. This instrument was linear within l-20/0 throughout the ranges used, permitted individual baseline setting for each peak, and was reproducible on repeated runs to within 23% (at least two integrations were done for each peak). It was used at maximal sensitivity settings, which gave 220 counts/ems, the area provided by 1.2-3 pg urease, depending on the staining intensity of the particular experiment. The model 542 Densicord has multiple response modes. The L mode corresponds to a per cent transmission response, while mode 5 corresponds to an absorbance response from 0 to 1.0 OD. Modes 6 to 12 provide intentionally progressive deviations from a strict logarithmic or absorbance response, and were of little use. The D-l mode is intended to provide a linear absorbance response from 0 to 2.0 OD, and modes D-2 and D-3 provide similar deviations from the D-l response. All response positions were analyzed with a series of Eastman Kodak neutral density filters mounted by taping at the edges to a strip of Mylar, after their optical densities were determined at 570 nm on a Beckman DU spectrophotometer. With spaces between the neutral density filters the reproducibility of baseline leveling was found to be excellent and the response time of the pen was good, covering 83% of a full-scale deflection in 1 sec. The correlation of scale reading with optical density for the pertinent response modes is shown in Fig. 1. In mode 5 the response was linear with absorbance from 0 to 0.85 OD, which occurs at 92% scale, after which the response deteriorates strikingly. In the L mode the response was essentially linear from 0 to 0.2 OD, and could be used to quantitate weak bands, thereafter multiplying by 0.525, the slope ratio relative to that of mode 5. The D-l setting was linear from 0.85 to 1.65 OD (again deteriorating above 92% scale), and when this line was extrapolated to the origin (shown by the dashed line) the slope was exactly one-half that of mode 5. At lower optical densities, however, the scale reading sagged far below linear response. A similar but even more striking pattern is seen in the D-3 mode, where the response is linear from 1.25 to 2.0 OD (again breaking down at 92% full scale), with a slope that is 2.3 times lower than that of mode 5.
392
WIIJiIAM
OPTICAL
N.
FlSIIBEIN
DENSITY
1. Plot of Photovolt 542 denaitometcr scale readings in various response modes, against actual absorbance of calibrated neutral density filters. Mode 5 is intended to provide a linear response with absorbance to 1.0 OD, and mode D-l, to 2.0 OD. Mode L is linear with % transmission. In all modes the response deteriorates markedly above 92% scale. See text for further details. FIG.
Thus a completely linear response with absorbance from 0 to 2.0 OD could be obtained in the following manner: all peaks were run in mode 5; each peak which exceeded 92.5% scale was rerun in mode D-l ; if the peak still exceeded 92.5% scale, it was rerun in D-3 mode. For such peaks the area was integrated up to 92% scale in the mode 5 trace, between 47% and 92% scale in the D-l trace, and between 82% and 92% scale in the D-3 trace. Addition of the third area X 2.3, and the second area X 2.0, to the first area then gave a total area based on a strictly linear response with absorbance. In practice, the D-l setting was only occasionally required, and the D-3 setting was not needed. For densitometry, an individual gel strip was cut from the slab, drained of excess water, centered on the glass plate of the carriage tray, and covered with a strip of Saran Wrap (adhering to the glass plate on all sides), making sure there were no air bubbles on either gel face. The whole was then drained of all excesswater and dried on the glass undersurface, placed in the carriage tray, and scanned from the bottom to the gel slit. Repeated runs could be carried out in this way without any evaporative
QUANTITATIVE
DENSITOMETRY
OF
PROTEIN
393
losses; and, provided the gel strips were carefully aligned so that the slit passed down the center of all bands, repeated chart runs were superimposable by transillumination, or virtually so, even when the gel strip was demounted and remounted. RESULTS
AND
DISCUSSION
As is not surprising for a noncovalently bound stain, the band intensity per unit, weight of protein varied from gel to gel. In terms of area units/ pg urease, the range encountered was 73-188. Since the integrator can, quantitate about 40 units with fair precision, the measurement limit was actually 0.2-0.5 pg urease; and the stated 1 pg minimum is a conservative estimate. The marked sensitivity of Coomassie blue as compared to amido black in the detection of urease is shown in Fig. 2. Two 5% gels were run under identical conditions in tandem chambers; each contained in sequential slots 5, 10, 20, 30, 40, 60, 80, and 100 pg urease. The left gel was stained with Coomassie blue and the right with amido black. Both were destained gradually to provide careful control against destaining of any bands. The urease preparation used for this comparison contained, in addition to the predominant a-band, a small amount of dimer, which appears as a faint
FIG. 2. Comparison of Coomns..ic blue (left) and amido black (right) staining of urease. Identical amounts of enzyme were placed in corrraponding slots of two 5% acrylamide gels, which were electrophoresed in tandem chambers for 6 hr at 3OOV, pH 8.5. From cenier to outer edge, rach gel contains 5, 10. 20, 30, 40, 60, 80. and 100 pg urcase in respective slots. The major component, a-urease, at 9 cm, is as strongly stained by Coomassie blue at the 5 pg level, as it is by amido black at the 40 pg level. The trace of urease dimer at 4.5 cm can be seen at the 10 pg level with Coomassie blue, and is barely visible at the 100 pg level with amido black.
394
WILLIAM
70 -
N.
FISHBEIN
56 pglpeak
6D-
c
” 30 xl
FIG.
11
IG
3. A (top)
: Triple
9
8 7 DISTANCE FROM ORIGIN
application
of series
6 (cml
of a-urease
5
standards
4
on a single
5%
QUANTITATIVE
DENSITOMETRY
OF
PROTEIN
395
band with slightly more than half the mobility of the a-band. The dimer is barely visible at the 100 pg level with amido black, whereas it can be seen at the 10 pg level with Coomassie blue. The 5 pg a-band in Coomassie blue is as strong as the 40 ,ug a-band in amido black. Thus the Coomassie blue stain is about an order of magnitude more sensitive for the detection of urease, as has been the case with other proteins (1,2). For densitometric quantitation, preparations of pure a-urease were used, and successive series of standards were electrophoresed in the same gel, so that the standard curves could be compared for different migration distances. An example is shown in Fig. 3A, where a 5% gel contained three series of 5.6-56 pg cy-urease standards which had migrated 4, 7, and 10 cm. It is evident that the band width increases with the quantity of protein, and with migration distance for a given quantity of protein. The bands in the third slot are distorted by an artifact in the gel, and this slot was not quantitated. The clarity and regularity of the background between bands is evident in Fig. 3B, which shows the densitometric traces of the first and last slots of Fig. 3A. The baseline was consistent throughout. the trace and there was little subjectivity involved in deciding on the areas to be integrated. To compare this with the usual situation in gel densitometry, one might examine the traces analyzed by Gorovsky et al. (3). The integrated areas from a series of four experiments were normalized to a value of 73 area units/pg, the lowest value obtained in the four gels. The results are shown in Fig. 4, grouped according to the migration dist.ance of each series of standards. In Fig. 4A, the values for standards which had migrated more than 9 cm from the origin are strictly linear through 55 ,ug urease, with a zero intercept. The close fit provided by standards from three different gels attests to the accuracy of sample application, the uniformity of staining across a single gel slab, and to the wide range over which Coomassie blue staining is proportional to protein weight under these conditions. The same straight line is therefore shown as the reference (solid line) in each of the other segments of Fig. 4. In Fig. 4B, standards which had migrated 7-8 cm fit the reference line perfectly well up to about 30 ,ug urease, beyond which some deviation below the line occurs, although it rarely exceeds 10%. However, in Fig, 4C, where the standards had migrated 4-5 cm, the deviation from the acrylamide gel stained with Coomassie blue. From left to right the slots contain 5.6, 11.2, 16.8, 22.4, 33.6, 44.8, and 56 pg a-urease applied in three series, with 2 hr electrophoresis at 3OOV, pH 8.5 between applications. No contaminant protein is detectable, and band width increases with migration distance as well as with the quantity of protein. Slot 3 was not quantitated because of the distortion artifact. B (bottom): Densitometric trace of first and last slots of the gel shown in A. The peaks are well separated by reproducible baselines over a lo-fold range of protein applied, and there is little subjectivity involved in selecting the areas for integration. The actual distance from O-100% scale on the densitometer is 4.5 in.
WILLIAM
N.
FISHBEIN
r/=7-Bcm
d=4-5cm
2500-
5
IO 15 20 25 30 35 40 45 50 55
5
IO 15 20 25 30 35 40 45 50 5
FIG. 4. Plot of densitometrie area versus urease applied (in rg) for four different migration distances from slot origin. Data are from four gels, each denoted by a separate symbol, and each gel had multiple applications of standards at different intervals of electrophoresis. In A, the area is proportional to protein quantity from 1 to 55 pg for bands that have migrated more than 9 cm. The stain intensity in different gels varied from 73 to 188 area units/pg, and all gels were normalized to the lowest value. The solid line is redrawn in each of the other sections of the figure as a reference. In B, bands migrating 7-8 cm show slight deviation below the reference line at protein levels above 30 pg. At shorter migration distances, in C and D, the deviations begin at lower protein levels and become progressively more marked. Thus the range of linearity varies directly with migration
QUANTITATlVE
DENSITOMETRY
OF
397
PROTEIN
reference line begins earlier, at about 20 /kg urease, and now amounts to a 20-257, error. Note, however, that the reference line still fits the values below 20 pg urease accurately. In Fig. 4D, where the migration distance was only 2.2 cm, the reference line fits the data only to 12 pg urease, and the deviation beyond this point was about 40%. Comparison of these four graphs leaves litt,le doubt. that migration distance, and hence barn1 compression within the gel, has a marked effect on the range over which linearity is followed. The only apparent explanation for this phenomenon is that the dye cannot fully penetrate and complex the compacted protein bands that are present’ after short migration distances. This would set a decided limit. on the value of band sharpness where quantitation rather than resolution is the prime oband hence compaction, is considerably jective. Since band sharpness, greater in cellulose acetate and disc electrophoresis than in acrylamide slabs, this is probably the reason that others have noted deviation of Coomassie blue staining from linearity at about 20 pg protein (1,2). For all bands migrating 9 cm or more, quantitation is readily provided for a I-50 pg range of protein, which should fully satisfy even the most critical user. In addition, the standard reference line would be satisfactory for quantitating all bands of 2 cm and greater migration, if no more than 12 pg of protein were applied to the slot. To my knowledge, the influence (or lack of influencej of migration distance on densitometric quantitation of protein bands has not. been evaluated for any other dye. It is possible that Coomassie blue is unique in staining behavior, because of its rigid platelike structure (1)) but there is no convincing published evidence that quantitation of protein by other dyes is, in fact, unaffected by migration distance, or its probable causative correlate, band compaction. To provide a 50-fold quantitation range regardless of migration distance, it is apparent that correction factors would be necessary. If we accept a lo-15% error for densitometric measurement (and I am most skeptical of claims to better precision than this), then a series of approximation lines may be developed to accommodate the deviating points for migration distances less than 9 cm. Such approximation lines are shown by the dashed lines in Fig. 4B-D ; by using their slopes relative to that of the standard reference line, and their intercepts with it, the necessary correction factors can be obtained. Figure 5A shows a plot of the intercept, I (in terms of pg protein), of approximation lines with the standard reference line, as related to the migration distance, d. A distance mations
(and band width). for the deviating
The points.
dashed
lines
show
the
best-fit
rectilinear
approxi-
WILLIAM
FIG. 5. A (left): stain, depending on the range extends migration. B (right) to that within, the linearity extends to in excess of this is
N.
FISHBEIN
Plot of maximum weight of protein that yields a proportional migration distance. The line is not straight above 9 cm, where to 50 gg protein, and there is no documentation below 2 cm : Fractional staining intensity of protein beyond, as compared range of linearity, as function of migration distance. At 10 cm 55 pg protein, the maximum amount tested, and any distance considered infinite, for the purposes of this graph.
straight line2 is obtained from which one can determine, for any migration distance, the maximum amount of urease that could be quantitated without correction. Similarly, the slope ratio, $, of approximation lines to the reference line is also a straight line* when plotted against the mi,gration distance (Fig. 5B). From these plots one can obtain the corrected weight (IV,) for a urease band anywhere on the gel from its nominal or uncorrected weight (WtL) : Where Id 2 W,, then W, = W,, and no correction is needed. Where Id < WV,, then W, = Id + (W, - Id)/+. The migration distance, d (in cm), of each band center from the origin must therefore be determined; in addition, since staining intensity varies ‘It is to be understood that the linearity is neither dictated by theory nor purely fortuitous. Acceptance of a 10-15% error permits some leeway in selecting approximation lines, and this was sufficient to permit the development of the rectilinear graph. The convenience of such a graph for interpolation and extrapolation outweighs any advantage of a statistical analysis at the level of accuracy involved here, although there is no evidence to verify extrapolation of the line below 2 cm migration distance. The graphs are readily convertible to simple linear equations for use with a small computer.
QUANTITATIVE
DENSITOMETRY
OF
PROTEIN
399
from gel to gel, at least one slot must contain a pure standard of known weight, to provide the area units/pg = F, for that gel. The nominal weight of any other band can then be determined as W, = area units/F. Alternatively, if migration distances are known, Fig. 5A permits a selection of the maximum loading dose that can be quantitated directly. It is disconcerting that so elaborate a procedure is required to provide acceptable densitometric quantitation, but the data allow of no simpler interpretation. Moreover, a number of addit,ional complexities must also be considered, especially if one is dealing with multiple proteins: since the standards were all evaluated on 5% gels, there is no guarantee that the correction factors for urease would hold for other gel strengths. Second, there is no guarantee that the correction factors for urease would apply t,o other proteins. Finally, since the color intensity per unit weight may vary from one protein to another, an accurate comparison of stain intensities would require electrophoresing a homogeneous form of each of the proteins to the same distance on the same gel, obviously not a readily feasible procedure. An attempt has been made to evaluate some of these problems by comparing urease with serum albumin and with thyroglobulin in paired 5% and 6% gels run under identical conditions in tandem chambers. A 1% increase in gel strength produces a marked decrease in mobility, especially for the higher weight proteins, and therefore provides a fair test of whether the correction factors based on the 5% gels are applicable to other gel strengths. The data in Table 1 give an affirmative answer in the case of urease. The stain intensity of urease on 5% and 6% gels, after correction for migration distance, showed an average deviation of 4% in experiment 486, and of less than 1% in experiment 484, whereas the corresponding deviations before distance correction were 21% and 11%. Other quantitations of urease by this procedure have also shown good agreement between 57, and 6% gel9 (9). This suggests that the distance correction for a given protein is relatively independent of gel strength. In experiment 486, serum albumin, electrophoresed at two concentrations along with urease, migrated more than 9 cm at both gel strengths, and was quantitated without distance correction. The two concentrations in a single gel show excellent agreement in terms of stain intensity per unit weight. Comparison between the 5% and 6% gels provides an acceptable average deviation of 7.5%; but this is much reduced if one compares on the basis of urease stain intensity as lOOoJ0, suggesting that the two gels have, in fact, not stained identically.3 It is also of interest that urease stains more intensely with Coomassie blue than does serum albumin, despite a 7-fold greater molecular weight. The very first com’ Earh
gel is stained
and
destained
in a separate
tray.
400
WILLIAM
N.
FISHBEIN
TABLE 1 Comparison of Coomassie Blue Staining Intensity of Urease, Bovine Serum Albumin (BSA), and Thyroglobulin at Different Gel Strengths, before and after Distance Correction
Gel strength (Expt. No.) 5%
(486)
6% (486)
5%
(484)
6% (484)
Protein Urease BSA BSA Urease BS.4 BSA Urease Thyroglobulin Urease Thyroglobulin
Pg 40 40 20 40 40 20 40
40 40
40
Stain intensity area units/rg
Migration distance, mm
Measured
Corrected
52 115
152 156.4
188 (156.4)
100
111
155.6 100
(155.6) 172
80 100
138.5 130.5 67.1 35.4 53.9 22.1
1138.5) (130.5) 86.8 64.9 86.2 88.8
32.5 94 94 52 30 36 17
‘$7~
81
81 76
100 75 100 103
For each experiment two gels were electrophoresed in tandem chambers for 3 hr at 300 V, pH 8.5. Procedures for staining, destaining, densitometry, and distance correction of the integrated areas are described in the text.
parison is thus sufficient to demonstrate significant variation in staining intensity from one protein to another. This is not surprising, and is hardly unique to Coomassie blue; no method of protein measurement, even in solution, is free of this problem. Thyroglobulin, which migrates more slowly than urease, is compared with it in experiment 484. For this protein, the distance-corrected stain intensit’ies in 5% and 65% gels show an average deviation of almost 16% (compared to 23% deviation for uncorrected data), thus raising the possibility that the correction faetora developed for urease may not be satisfactory for other proteins. The correction factors are being stretched to their limits here, since the migration of thyroglobulin in the 6% gel was only 1.7 cm, and this could be responsible for the deviation. We must leave the more critical evaluation of the densitometry of multiple types of proteins to those who are directly concerned with such studies. The method detailed above will clearly provide acceptable quantitative data at very low concentrations for isologous forms of a single protein. The current increase of interest in conformeric and polymeric forms of enzymes and other proteins, which in many cases are first encountered and elucidated by gel electrophoresis, suggests that accurate densitometric quantitation of such forms will become increasingly valuable. More than twenty enzymes are known to exhibit polymer series (lo), in addition to those of nonenzymic proteins. The use
QUANTITATIVE
DENSITOMETRY
OF
401
PROTEIN
of gel electrophoresis to evaluate the kinetic and equilibrium distribution of multiple forms of a protein should be of interest to all who wish to study macromolecular association-dissociation phenomena. SUMMARY
A critical study has been made of quantitative densitometry with Coomassie blue in acrylamide gel slabs, using successive applications of a pure protein, a-urease, to provide peaks well separated by reproducible baselines, a densitometer calibrated for linear response with optical density, and repet’itive digital area integration. Staining was proportional to protein weight over a range that was markedly dependent upon migration distance in the gel. For bands that had migrated more than 9 cm, linearity was followed over a range of l-55 /lg. At shorter migration distances the range of linearity progressively contracted, until at 2 cm migration it encompassed only 1-12 pg. This pattern suggests that progressive compaction of the protein bands at shorter migration distances is the responsible factor, preventing the stoichiometric uptake of dye molecules once a critical protein concentration has been exceeded. Accepting a lO-15% error in densitomctry, the deviations could be corrected by application of an empirically derived set of factors, so that, a l-50 ,ug range could be quantitated for any distance beyond 2 cm from the slot origin. The correction factors, although derived for 5% acrylamide gels, were also applicable to other gel strengths. REFERENCES 1. FAZEKAS
DE ST.
GROTH.
S., WEBSTER,
R. G., AND
DATYNER,
A., Biochim.
Biophys.
Acta 71, 377 (1963). 2. CHRAMBACH, A., REISFELD, R. A., WYCKOFF, M., AND ZACCARI, J., Anal. Biochem. 20, 150 (1967). 3. GOROVSKY, M. A., CARLSON, Ii., AND ROSENBAUM, J. L., Anal. Biochem. 35, 359 (1970). 4. FISIIBEIN, W. N., NAUARAJAN, K., .~ND SCURZI, W., J. Biol. Chem. 245, 5985 (1970). 5. FISHBEIN, W. PIT., SPEARS, C. L., AND SCURZI, W., Nature 223, 191 (1969). 6. FISIIBEIN, W. N., Ann. N. I’. Ad. Sci. 147, 857 (1969). ‘7. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL. R. J., J. Biol. Chrm. 193, 265 (1951). 8. Ortec Application Note AN32, “Techniques for High Resolution Electrophoresis,” Oak Ridge, 1970, p. 15. 9. FISHBEIN, W. N.. ASII NAQARAJAN, K., Arch. Biochem. Biophys. 144, 700 (1971). 10. MARRERT. C. L.. ASD WHITT, G. S., Expetientia 24, 977 (1968).
46,
BIOCHEMISTRY
Quantitative
388401
(1972)
Acrylamide
Gel
Slabs
WILLIAM Biochemistry
Branch,
of l-50
Densitometry
Armed
Forces
with
rug Protein
Coomassie
in
Blue’
N. FISHBEIN Institute
Received
April
of
Pathology,
Washington,
D. C. .80306
5, 1971
Polyacrylamide gel electrophoresis is the most sensit’ive technique presently available for separating proteins, and Coomassie blue is the most sensitive protein stain available (1). Together they are regularly used to separate and identify microgram quantities of proteins. Unfortunately, Coomassie blue has been reported to follow Beer’s law over only a narrow concentration range (1,2), and other less sensitive dyes have been suggest,ed for quantitation over a wider range (3). However, this involves duplicating all runs, since the great sensitivity of Coomassie blue is not readily forsaken, or else working at lo-fold greater protein concentrations, if feasible. It would obviously be an advantage to use Coomassie blue both for identification and for quantitation over a reasonable range, and this has impelled the present study. Of more general concern is the lack of convincing evidence as to the accuracy of quantitative dye densitometry, despite the widespread use of this procedure. The major sources of error in quantitative densitometry may be summarized as follows: (a) variation in the amount of dye bound from one protein to another, (b) baseline selection and peak-splitting procedures whenever there is no baseline segment between peaks, (c) nonlinearity in the detector and/or pen response to absorbance, (d) integrator inaccuracy, especially for small areas. The first problem is absent if one deals with a family of isologous proteins, i.e., a single protein which assumes multiple forms. The most readily diagnosed case of such a family is an enzyme displaying non’ This work was supported in part by grant AM-10900 from the United States Public Health Service and in part by a research contract, Project 3AO61102B71P-06, from the United States Army Medical Research and Development Command. The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. 388 @ 1972 by Academic
Press.
Inc.
QUANTITATIVE
DENSITOMEX’RY
OF
PROTEIN
389
genetic isozymes, where the same primary structure in all forms would dictate equivalent dye-binding for all bands. Thus the quantitation of a series of enzyme polymers should offer a most favorable situation for densitometry. Of specific interest here is the enzyme urease, which can be prepared in a form homogeneous by both electrophoresis and ultracentrifugation (4), and from which can be produced a polymer series, as well as other nongenetic isozymes (5,6). Since the bands are well separated on electrophoresis, there is no difficulty in baseline estimation and no need for peak splitting; so t’he second source of error is also absent. The third and fourth objections were removed by standardizing the densit’ometer with a series of neutral density filters, and by the use of repetitive digital integration. Using therefore the most favorable possible conditions for densitometric accuracy, we can test whether the dye uptake is proportional to the amount of protein applied, and more rigorously, we can test the most obvious question to which we have seen no answer in the literature: is the dye uptake of a protein independent of its migration distance, and hence of its band width? As will be seen below, the answer to this latter question is a depressing “no” in the case of Coomassie blue, although ureas@ can nevertheless be quantitated after suitable corrections. MATERIALS
AND
METHODS
Chemicals Pure ol-urease, the monomer with molecular weight 480,000, was prepared from jackbean meal as previously described (4) and the protein content was determined with the Folin-phenol reagent (7). Coomassieblue was obtained from Mann Research Laboratories (New York, N. Y.) and prepared as 1% stock solutions in absolute ethanol. Thyroglobulin was also obtained from Mann Research Laboratories; and 3X crystallized bovine serum albumin was obtained from Pentex (Kankakee, Ill.). Stock solutions of 60% trichloroacetic acid were prepared in water and diluted 1:5 as needed. Electrophoresis Vertical .gel slab electrophoresis assemblies (EC Apparatus Corp., Philadelphia, Pa.) were cooled by circulating refrigerated water to maintain a gel temperature of 10” throughout the run, A mixture of 95% acrylamide/5% methylene bisacrylamide was used in all experiments, and was weighed in an analytical balance to obtain 5% or 6% acrylamide gels, the former being used for all standard quantitations. Ammonium
390
WILLIAM
N.
FISHBEIN
persulfate and N,N,N’,N’-tetramethylethylenediamine were used to catalyze polymerization (6)) and all gels were prerun for at least 1 hr at 300 V before sample application. The same buffer was used for gel preparation and in the tank reservoirs: 0.05 M Tris-acetate, pH 8.5, with 1 miU EDTA ; buffer reservoirs were continuously pump-exchanged throughout all runs. Protein solutions were weighted by addition of an equal volume of 50% sucrose in water, and were applied with Hamilton syringes, keeping the needle tip at the extreme upper lip of each slot opening. With the electrophoresis assembly illuminated from behind, the refraction lines of the sucrose solution were readily observed, and volumes of l-60 ~1 could be applied in a slot with high accuracy. In many experiments, after a l-2 hr electrophoresis, the voltage and buffer pumps were turned off, a second series of samples was applied, and further electrophoresis was then carried out. This could be repeated once or twice more, depending on the electrophoresis intervals selected. Staining
and Photography
The gels were fixed and stained by gentle rocking overnight in plastic boxes containing a solution prepared by diluting 1% Coomassie blue in absolute ethanol 20-fold with 12% trichloroacetic acid. Protein staining was not significantly increased on prolonging the staining period beyond 12 hr (up to 72 hr), but 4-6 hr was definitely insufficient time for full staining. After rinsing out the dye with distilled water, the gel was removed a;ld the tray was rinsed several times with methanol and then water before replacing the gel for overnight destaining in 12% trichloroacetic acid. A second overnight destaining was normally sufficient to completely clear the gel background. After destaining, the gels were equilibrated with water, and photographed with an MP-3 Polaroid system, using a pink-tinted transilluminator (Cole-Parmer, Chicago, Ill.) ; no filter was necessary. Other methods of staining and destaining with Coomassie blue were found wanting when visually compared to that above, using duplicate samples and a split-gel technique. Electrophoretic destaining was variable and uneven. The use of aqueous Coomassie blue after prior gel fixing (8) was definitely less sensitive, even when much higher concentrations of the dye were used. Hydrochloric acid (1 N) was not effective in destaining the gel background, although free dye was more soluble in this acid than in trichloroacetic acid. On the other hand, destaining by any method was extremely difficult after overnight storage in 6% aqueous acetic acid. Aqueous stock solutions of Coomassie blue (2) gradually decomposed to yield a gummy residue; the reaction was accelerated in trichloroacetic acid. and retarded in ethanol.
QUANTITATIVE
DENSITOMETRY
Densitometry
OF
PROTEIN
391
and Integration
A Photovolt model 542 Densicord was equipped with a fine resolution slit (0.1 X 6 mm), starch gel carriage geared to 0.5 in./min travel, chart expansion accessory set at 2.411 expansion, and a 570 nm filter, which gave maximum absorption for Coomassie blue stained proteins. The Photovolt disc integrat,or was found to be erratic and unreliable, and the chart strips were instead integrated with the Technicon model AAG digital integrator in the linear mode, with the chart speed set at 4.5 in./ min. This instrument was linear within l-20/0 throughout the ranges used, permitted individual baseline setting for each peak, and was reproducible on repeated runs to within 23% (at least two integrations were done for each peak). It was used at maximal sensitivity settings, which gave 220 counts/ems, the area provided by 1.2-3 pg urease, depending on the staining intensity of the particular experiment. The model 542 Densicord has multiple response modes. The L mode corresponds to a per cent transmission response, while mode 5 corresponds to an absorbance response from 0 to 1.0 OD. Modes 6 to 12 provide intentionally progressive deviations from a strict logarithmic or absorbance response, and were of little use. The D-l mode is intended to provide a linear absorbance response from 0 to 2.0 OD, and modes D-2 and D-3 provide similar deviations from the D-l response. All response positions were analyzed with a series of Eastman Kodak neutral density filters mounted by taping at the edges to a strip of Mylar, after their optical densities were determined at 570 nm on a Beckman DU spectrophotometer. With spaces between the neutral density filters the reproducibility of baseline leveling was found to be excellent and the response time of the pen was good, covering 83% of a full-scale deflection in 1 sec. The correlation of scale reading with optical density for the pertinent response modes is shown in Fig. 1. In mode 5 the response was linear with absorbance from 0 to 0.85 OD, which occurs at 92% scale, after which the response deteriorates strikingly. In the L mode the response was essentially linear from 0 to 0.2 OD, and could be used to quantitate weak bands, thereafter multiplying by 0.525, the slope ratio relative to that of mode 5. The D-l setting was linear from 0.85 to 1.65 OD (again deteriorating above 92% scale), and when this line was extrapolated to the origin (shown by the dashed line) the slope was exactly one-half that of mode 5. At lower optical densities, however, the scale reading sagged far below linear response. A similar but even more striking pattern is seen in the D-3 mode, where the response is linear from 1.25 to 2.0 OD (again breaking down at 92% full scale), with a slope that is 2.3 times lower than that of mode 5.
392
WIIJiIAM
OPTICAL
N.
FlSIIBEIN
DENSITY
1. Plot of Photovolt 542 denaitometcr scale readings in various response modes, against actual absorbance of calibrated neutral density filters. Mode 5 is intended to provide a linear response with absorbance to 1.0 OD, and mode D-l, to 2.0 OD. Mode L is linear with % transmission. In all modes the response deteriorates markedly above 92% scale. See text for further details. FIG.
Thus a completely linear response with absorbance from 0 to 2.0 OD could be obtained in the following manner: all peaks were run in mode 5; each peak which exceeded 92.5% scale was rerun in mode D-l ; if the peak still exceeded 92.5% scale, it was rerun in D-3 mode. For such peaks the area was integrated up to 92% scale in the mode 5 trace, between 47% and 92% scale in the D-l trace, and between 82% and 92% scale in the D-3 trace. Addition of the third area X 2.3, and the second area X 2.0, to the first area then gave a total area based on a strictly linear response with absorbance. In practice, the D-l setting was only occasionally required, and the D-3 setting was not needed. For densitometry, an individual gel strip was cut from the slab, drained of excess water, centered on the glass plate of the carriage tray, and covered with a strip of Saran Wrap (adhering to the glass plate on all sides), making sure there were no air bubbles on either gel face. The whole was then drained of all excesswater and dried on the glass undersurface, placed in the carriage tray, and scanned from the bottom to the gel slit. Repeated runs could be carried out in this way without any evaporative
QUANTITATIVE
DENSITOMETRY
OF
PROTEIN
393
losses; and, provided the gel strips were carefully aligned so that the slit passed down the center of all bands, repeated chart runs were superimposable by transillumination, or virtually so, even when the gel strip was demounted and remounted. RESULTS
AND
DISCUSSION
As is not surprising for a noncovalently bound stain, the band intensity per unit, weight of protein varied from gel to gel. In terms of area units/ pg urease, the range encountered was 73-188. Since the integrator can, quantitate about 40 units with fair precision, the measurement limit was actually 0.2-0.5 pg urease; and the stated 1 pg minimum is a conservative estimate. The marked sensitivity of Coomassie blue as compared to amido black in the detection of urease is shown in Fig. 2. Two 5% gels were run under identical conditions in tandem chambers; each contained in sequential slots 5, 10, 20, 30, 40, 60, 80, and 100 pg urease. The left gel was stained with Coomassie blue and the right with amido black. Both were destained gradually to provide careful control against destaining of any bands. The urease preparation used for this comparison contained, in addition to the predominant a-band, a small amount of dimer, which appears as a faint
FIG. 2. Comparison of Coomns..ic blue (left) and amido black (right) staining of urease. Identical amounts of enzyme were placed in corrraponding slots of two 5% acrylamide gels, which were electrophoresed in tandem chambers for 6 hr at 3OOV, pH 8.5. From cenier to outer edge, rach gel contains 5, 10. 20, 30, 40, 60, 80. and 100 pg urcase in respective slots. The major component, a-urease, at 9 cm, is as strongly stained by Coomassie blue at the 5 pg level, as it is by amido black at the 40 pg level. The trace of urease dimer at 4.5 cm can be seen at the 10 pg level with Coomassie blue, and is barely visible at the 100 pg level with amido black.
394
WILLIAM
70 -
N.
FISHBEIN
56 pglpeak
6D-
c
” 30 xl
FIG.
11
IG
3. A (top)
: Triple
9
8 7 DISTANCE FROM ORIGIN
application
of series
6 (cml
of a-urease
5
standards
4
on a single
5%
QUANTITATIVE
DENSITOMETRY
OF
PROTEIN
395
band with slightly more than half the mobility of the a-band. The dimer is barely visible at the 100 pg level with amido black, whereas it can be seen at the 10 pg level with Coomassie blue. The 5 pg a-band in Coomassie blue is as strong as the 40 ,ug a-band in amido black. Thus the Coomassie blue stain is about an order of magnitude more sensitive for the detection of urease, as has been the case with other proteins (1,2). For densitometric quantitation, preparations of pure a-urease were used, and successive series of standards were electrophoresed in the same gel, so that the standard curves could be compared for different migration distances. An example is shown in Fig. 3A, where a 5% gel contained three series of 5.6-56 pg cy-urease standards which had migrated 4, 7, and 10 cm. It is evident that the band width increases with the quantity of protein, and with migration distance for a given quantity of protein. The bands in the third slot are distorted by an artifact in the gel, and this slot was not quantitated. The clarity and regularity of the background between bands is evident in Fig. 3B, which shows the densitometric traces of the first and last slots of Fig. 3A. The baseline was consistent throughout. the trace and there was little subjectivity involved in deciding on the areas to be integrated. To compare this with the usual situation in gel densitometry, one might examine the traces analyzed by Gorovsky et al. (3). The integrated areas from a series of four experiments were normalized to a value of 73 area units/pg, the lowest value obtained in the four gels. The results are shown in Fig. 4, grouped according to the migration dist.ance of each series of standards. In Fig. 4A, the values for standards which had migrated more than 9 cm from the origin are strictly linear through 55 ,ug urease, with a zero intercept. The close fit provided by standards from three different gels attests to the accuracy of sample application, the uniformity of staining across a single gel slab, and to the wide range over which Coomassie blue staining is proportional to protein weight under these conditions. The same straight line is therefore shown as the reference (solid line) in each of the other segments of Fig. 4. In Fig. 4B, standards which had migrated 7-8 cm fit the reference line perfectly well up to about 30 ,ug urease, beyond which some deviation below the line occurs, although it rarely exceeds 10%. However, in Fig, 4C, where the standards had migrated 4-5 cm, the deviation from the acrylamide gel stained with Coomassie blue. From left to right the slots contain 5.6, 11.2, 16.8, 22.4, 33.6, 44.8, and 56 pg a-urease applied in three series, with 2 hr electrophoresis at 3OOV, pH 8.5 between applications. No contaminant protein is detectable, and band width increases with migration distance as well as with the quantity of protein. Slot 3 was not quantitated because of the distortion artifact. B (bottom): Densitometric trace of first and last slots of the gel shown in A. The peaks are well separated by reproducible baselines over a lo-fold range of protein applied, and there is little subjectivity involved in selecting the areas for integration. The actual distance from O-100% scale on the densitometer is 4.5 in.
WILLIAM
N.
FISHBEIN
r/=7-Bcm
d=4-5cm
2500-
5
IO 15 20 25 30 35 40 45 50 55
5
IO 15 20 25 30 35 40 45 50 5
FIG. 4. Plot of densitometrie area versus urease applied (in rg) for four different migration distances from slot origin. Data are from four gels, each denoted by a separate symbol, and each gel had multiple applications of standards at different intervals of electrophoresis. In A, the area is proportional to protein quantity from 1 to 55 pg for bands that have migrated more than 9 cm. The stain intensity in different gels varied from 73 to 188 area units/pg, and all gels were normalized to the lowest value. The solid line is redrawn in each of the other sections of the figure as a reference. In B, bands migrating 7-8 cm show slight deviation below the reference line at protein levels above 30 pg. At shorter migration distances, in C and D, the deviations begin at lower protein levels and become progressively more marked. Thus the range of linearity varies directly with migration
QUANTITATlVE
DENSITOMETRY
OF
397
PROTEIN
reference line begins earlier, at about 20 /kg urease, and now amounts to a 20-257, error. Note, however, that the reference line still fits the values below 20 pg urease accurately. In Fig. 4D, where the migration distance was only 2.2 cm, the reference line fits the data only to 12 pg urease, and the deviation beyond this point was about 40%. Comparison of these four graphs leaves litt,le doubt. that migration distance, and hence barn1 compression within the gel, has a marked effect on the range over which linearity is followed. The only apparent explanation for this phenomenon is that the dye cannot fully penetrate and complex the compacted protein bands that are present’ after short migration distances. This would set a decided limit. on the value of band sharpness where quantitation rather than resolution is the prime oband hence compaction, is considerably jective. Since band sharpness, greater in cellulose acetate and disc electrophoresis than in acrylamide slabs, this is probably the reason that others have noted deviation of Coomassie blue staining from linearity at about 20 pg protein (1,2). For all bands migrating 9 cm or more, quantitation is readily provided for a I-50 pg range of protein, which should fully satisfy even the most critical user. In addition, the standard reference line would be satisfactory for quantitating all bands of 2 cm and greater migration, if no more than 12 pg of protein were applied to the slot. To my knowledge, the influence (or lack of influencej of migration distance on densitometric quantitation of protein bands has not. been evaluated for any other dye. It is possible that Coomassie blue is unique in staining behavior, because of its rigid platelike structure (1)) but there is no convincing published evidence that quantitation of protein by other dyes is, in fact, unaffected by migration distance, or its probable causative correlate, band compaction. To provide a 50-fold quantitation range regardless of migration distance, it is apparent that correction factors would be necessary. If we accept a lo-15% error for densitometric measurement (and I am most skeptical of claims to better precision than this), then a series of approximation lines may be developed to accommodate the deviating points for migration distances less than 9 cm. Such approximation lines are shown by the dashed lines in Fig. 4B-D ; by using their slopes relative to that of the standard reference line, and their intercepts with it, the necessary correction factors can be obtained. Figure 5A shows a plot of the intercept, I (in terms of pg protein), of approximation lines with the standard reference line, as related to the migration distance, d. A distance mations
(and band width). for the deviating
The points.
dashed
lines
show
the
best-fit
rectilinear
approxi-
WILLIAM
FIG. 5. A (left): stain, depending on the range extends migration. B (right) to that within, the linearity extends to in excess of this is
N.
FISHBEIN
Plot of maximum weight of protein that yields a proportional migration distance. The line is not straight above 9 cm, where to 50 gg protein, and there is no documentation below 2 cm : Fractional staining intensity of protein beyond, as compared range of linearity, as function of migration distance. At 10 cm 55 pg protein, the maximum amount tested, and any distance considered infinite, for the purposes of this graph.
straight line2 is obtained from which one can determine, for any migration distance, the maximum amount of urease that could be quantitated without correction. Similarly, the slope ratio, $, of approximation lines to the reference line is also a straight line* when plotted against the mi,gration distance (Fig. 5B). From these plots one can obtain the corrected weight (IV,) for a urease band anywhere on the gel from its nominal or uncorrected weight (WtL) : Where Id 2 W,, then W, = W,, and no correction is needed. Where Id < WV,, then W, = Id + (W, - Id)/+. The migration distance, d (in cm), of each band center from the origin must therefore be determined; in addition, since staining intensity varies ‘It is to be understood that the linearity is neither dictated by theory nor purely fortuitous. Acceptance of a 10-15% error permits some leeway in selecting approximation lines, and this was sufficient to permit the development of the rectilinear graph. The convenience of such a graph for interpolation and extrapolation outweighs any advantage of a statistical analysis at the level of accuracy involved here, although there is no evidence to verify extrapolation of the line below 2 cm migration distance. The graphs are readily convertible to simple linear equations for use with a small computer.
QUANTITATIVE
DENSITOMETRY
OF
PROTEIN
399
from gel to gel, at least one slot must contain a pure standard of known weight, to provide the area units/pg = F, for that gel. The nominal weight of any other band can then be determined as W, = area units/F. Alternatively, if migration distances are known, Fig. 5A permits a selection of the maximum loading dose that can be quantitated directly. It is disconcerting that so elaborate a procedure is required to provide acceptable densitometric quantitation, but the data allow of no simpler interpretation. Moreover, a number of addit,ional complexities must also be considered, especially if one is dealing with multiple proteins: since the standards were all evaluated on 5% gels, there is no guarantee that the correction factors for urease would hold for other gel strengths. Second, there is no guarantee that the correction factors for urease would apply t,o other proteins. Finally, since the color intensity per unit weight may vary from one protein to another, an accurate comparison of stain intensities would require electrophoresing a homogeneous form of each of the proteins to the same distance on the same gel, obviously not a readily feasible procedure. An attempt has been made to evaluate some of these problems by comparing urease with serum albumin and with thyroglobulin in paired 5% and 6% gels run under identical conditions in tandem chambers. A 1% increase in gel strength produces a marked decrease in mobility, especially for the higher weight proteins, and therefore provides a fair test of whether the correction factors based on the 5% gels are applicable to other gel strengths. The data in Table 1 give an affirmative answer in the case of urease. The stain intensity of urease on 5% and 6% gels, after correction for migration distance, showed an average deviation of 4% in experiment 486, and of less than 1% in experiment 484, whereas the corresponding deviations before distance correction were 21% and 11%. Other quantitations of urease by this procedure have also shown good agreement between 57, and 6% gel9 (9). This suggests that the distance correction for a given protein is relatively independent of gel strength. In experiment 486, serum albumin, electrophoresed at two concentrations along with urease, migrated more than 9 cm at both gel strengths, and was quantitated without distance correction. The two concentrations in a single gel show excellent agreement in terms of stain intensity per unit weight. Comparison between the 5% and 6% gels provides an acceptable average deviation of 7.5%; but this is much reduced if one compares on the basis of urease stain intensity as lOOoJ0, suggesting that the two gels have, in fact, not stained identically.3 It is also of interest that urease stains more intensely with Coomassie blue than does serum albumin, despite a 7-fold greater molecular weight. The very first com’ Earh
gel is stained
and
destained
in a separate
tray.
400
WILLIAM
N.
FISHBEIN
TABLE 1 Comparison of Coomassie Blue Staining Intensity of Urease, Bovine Serum Albumin (BSA), and Thyroglobulin at Different Gel Strengths, before and after Distance Correction
Gel strength (Expt. No.) 5%
(486)
6% (486)
5%
(484)
6% (484)
Protein Urease BSA BSA Urease BS.4 BSA Urease Thyroglobulin Urease Thyroglobulin
Pg 40 40 20 40 40 20 40
40 40
40
Stain intensity area units/rg
Migration distance, mm
Measured
Corrected
52 115
152 156.4
188 (156.4)
100
111
155.6 100
(155.6) 172
80 100
138.5 130.5 67.1 35.4 53.9 22.1
1138.5) (130.5) 86.8 64.9 86.2 88.8
32.5 94 94 52 30 36 17
‘$7~
81
81 76
100 75 100 103
For each experiment two gels were electrophoresed in tandem chambers for 3 hr at 300 V, pH 8.5. Procedures for staining, destaining, densitometry, and distance correction of the integrated areas are described in the text.
parison is thus sufficient to demonstrate significant variation in staining intensity from one protein to another. This is not surprising, and is hardly unique to Coomassie blue; no method of protein measurement, even in solution, is free of this problem. Thyroglobulin, which migrates more slowly than urease, is compared with it in experiment 484. For this protein, the distance-corrected stain intensit’ies in 5% and 65% gels show an average deviation of almost 16% (compared to 23% deviation for uncorrected data), thus raising the possibility that the correction faetora developed for urease may not be satisfactory for other proteins. The correction factors are being stretched to their limits here, since the migration of thyroglobulin in the 6% gel was only 1.7 cm, and this could be responsible for the deviation. We must leave the more critical evaluation of the densitometry of multiple types of proteins to those who are directly concerned with such studies. The method detailed above will clearly provide acceptable quantitative data at very low concentrations for isologous forms of a single protein. The current increase of interest in conformeric and polymeric forms of enzymes and other proteins, which in many cases are first encountered and elucidated by gel electrophoresis, suggests that accurate densitometric quantitation of such forms will become increasingly valuable. More than twenty enzymes are known to exhibit polymer series (lo), in addition to those of nonenzymic proteins. The use
QUANTITATIVE
DENSITOMETRY
OF
401
PROTEIN
of gel electrophoresis to evaluate the kinetic and equilibrium distribution of multiple forms of a protein should be of interest to all who wish to study macromolecular association-dissociation phenomena. SUMMARY
A critical study has been made of quantitative densitometry with Coomassie blue in acrylamide gel slabs, using successive applications of a pure protein, a-urease, to provide peaks well separated by reproducible baselines, a densitometer calibrated for linear response with optical density, and repet’itive digital area integration. Staining was proportional to protein weight over a range that was markedly dependent upon migration distance in the gel. For bands that had migrated more than 9 cm, linearity was followed over a range of l-55 /lg. At shorter migration distances the range of linearity progressively contracted, until at 2 cm migration it encompassed only 1-12 pg. This pattern suggests that progressive compaction of the protein bands at shorter migration distances is the responsible factor, preventing the stoichiometric uptake of dye molecules once a critical protein concentration has been exceeded. Accepting a lO-15% error in densitomctry, the deviations could be corrected by application of an empirically derived set of factors, so that, a l-50 ,ug range could be quantitated for any distance beyond 2 cm from the slot origin. The correction factors, although derived for 5% acrylamide gels, were also applicable to other gel strengths. REFERENCES 1. FAZEKAS
DE ST.
GROTH.
S., WEBSTER,
R. G., AND
DATYNER,
A., Biochim.
Biophys.
Acta 71, 377 (1963). 2. CHRAMBACH, A., REISFELD, R. A., WYCKOFF, M., AND ZACCARI, J., Anal. Biochem. 20, 150 (1967). 3. GOROVSKY, M. A., CARLSON, Ii., AND ROSENBAUM, J. L., Anal. Biochem. 35, 359 (1970). 4. FISIIBEIN, W. N., NAUARAJAN, K., .~ND SCURZI, W., J. Biol. Chem. 245, 5985 (1970). 5. FISHBEIN, W. PIT., SPEARS, C. L., AND SCURZI, W., Nature 223, 191 (1969). 6. FISIIBEIN, W. N., Ann. N. I’. Ad. Sci. 147, 857 (1969). ‘7. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL. R. J., J. Biol. Chrm. 193, 265 (1951). 8. Ortec Application Note AN32, “Techniques for High Resolution Electrophoresis,” Oak Ridge, 1970, p. 15. 9. FISHBEIN, W. N.. ASII NAQARAJAN, K., Arch. Biochem. Biophys. 144, 700 (1971). 10. MARRERT. C. L.. ASD WHITT, G. S., Expetientia 24, 977 (1968).