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Some quantitative aspects of disc electrophoresis
570 BBA
SHORT COMMUNICATIONS
33121
Some quantitative aspects of disc
electrophoresis
Although the disc electrophoretic technique of ORNSTEIN1 and DAVIS2 has become widely accepted as a qualitative tool for the resolution of proteins, quantitative data on the separated protein components is sparse a,4. While investigating the disc electrophoretic patterns of serum lipoproteins of several species, an anomaly in the peak area of the low density lipoproteins at different gel concentrations was observed a. In order to clarify this, we have extended our investigation to some of the quantitative aspects of the disc electrophoretic method using well-characterized proteins such as egg ovalbumin and hmnan serum albumin. We report here on factors such as gel concentration, penetration distances of protein into the main gel and sample quantities, which we observed to have a profound influence on densitometer peak areas. The equipment used in this study was the Model Ie Disc Electrophoresis Unit and the Model E Microdensitometer (Canalco, Rockville, Md.). Egg ovalbunfin (5 times crystallized), human serum albumin grade I I I (Sigma Chemical Co., St. Louis, Mo.) and Amidoblack IoB (Merck AG, Darmstadt, Germany) were the proteins and dye used in this report. The disc electrophoretic procedure was the same as Method C of NARAYAN, NARAYANAND KUM~IEROWa. Volumes of 1oo #1 were loaded for all ovalbumin quantities, except for the I6o-#g samples where 2o0 ffl were used. Unless otherwise stated, the main gel was z l inch long and the spacer gel was ½ inch long. The electrophoresis was terminated when the tracking dye (Bromphenol Blue) penetrated ~ inch into the main gel, except for the penetration studies where different distances of penetration were employed. After electrophoresis, the gels were stained in a 7-5% aq. acetic acid solution containing i ~Yo Amidoblack dye. Staining was allowed to proceed for ~-2 h. Staining times up to 8 h did not affect the areas under the densitometric pattern for the same sample size. Destaining was accomplished by repeatedly washing the gels over a period of 2- 4 days with a 7.5 % aq. acetic acid solution. Electrophoretic destaining, performed immediately after the staining time, gave tile same results as the washing procedure. The entire area under the densitometric pattern was used in obtaining the peak area and will henceforth be referred to as the total peak area. The peak areas were determined by counting the number of integrator pips under each peak a. The microdensitometer, with a tungsten light source, was fitted with a Wratten 24 gelatin filter and was checked by using neutral density filters. The integrator of the densitometer was also checked for accuracy by determining the nmnber of pips per I2 inch on chart paper at different absorbance settings, ranging from o.z to 4.I, using either the control knob on tile densitometer alone or in conjunction with the neutral density filters. It was observed that the integrator response was linear up to an absorbance of 3.0. The results indicated that there was a linear relationship in the total peak area with increasing amounts of protein only up to I5 #g of protein (Table I). With as little as lO/~g of ovalbumin, the maximum absorbance observed on tile densitometer was already ~.2 and served to emphasize the great sensitivity of tile technique. Samples containing as little as I #g of protein were detected visually as well as by the densitometer. Ovalbumin resolved into one main band and 3 4 minor bands. The minor bands Biochim. Bioph),s. Acla, 168 (1968) 57° 572
571
SHORT COMMUNICATIONS TABLE
I
TOTAL PEAK AREA WITH INCREASING AMOUNTS OF OVALBUMIN The concentration
o f m a i n g e l u s e d w a s 7.5 % .
Protein* (l~g)
Area N u m b e r of (cm 2 × 2.5) ** determinations
5 io 15 20 4° 80 16o
0.75 5.1 = lO. 3 ~= 14.7 ~= I8.4 ± 26.0 i 38.5 ± 5 °.6 =
0.8 I.O 1.3 0.9 2.t 1.9 3.9
2 8 9 5 9 18 7 8
* B o t h d y e a n d p r o t e i n a r e u n c o r r e c t e d f o r m o i s t u r e c o n t e n t i n all t h e T a b l e s . T h e m o i s t u r e c o n t e n t o f t h e o v a l b u m i n a n d A m i d o b l a c k i o B d y e w a s 8.2 % a n d 9 . 4 % , r e s p e c t i v e l y . *" V a l u e s g i v e n a r e t h e m e a n -k S . D .
TABLE
II
TOTAL PEAK AREA WITH INCREASING SAMPLE PENETRATION INTO THE MAIN GEL The concentration
Penetration (inch)
o f m a i n gel u s e d w a s 7 . 5 % .
Area (cm 2 × 2.5)* 40 I~g protein I6o itg protein
~1 16
I~ Ii~6
t4"° 23"7 25.3 29-7
27"3 5° 57 68.3
* A r e a s a r e a v e r a g e s o f 3 g e l s e a c h e x c e p t f o r t h e 11~ a n d i{t i n c h v a l u e s f o r t h e i 6 o - p g samples which are single determinations.
became increasingly less visible below 4 °/~g of protein sample. The total peak area became larger as the protein was allowed to penetrate increasing distances into the main gel. The total area more than doubled for each sample quantity depending on the degree of penetration into the main gel (Table II). Because of varying degrees of sample penetration at different gel concentrations, it was necessary to migrate the protein rather than the tracking dye to a preset distance. Since ovalbumin does not bind the tracking dye, human serum albumin, which appears as a purple band behind the tracking dye, was used in these experiments (Table III). As the gel concentration was increased from 3.75 to 7.5%, the total peak area increased 65 and 95%, respectively, when 4o and 16o/~g of protein were used. Increasing the gel concentration above 7.5 % had little effect, at least in the case of the small protein, human serum albumin. With large molecular weight substances, such as low density lipoproteins of human serum (mol. wt. 3.2" IO6), increases in gel concentration beyond 3.75 % caused compression rather than spreading and therefore gave rise to lower peak areas 5. The results of this study indicated the importance of two factors, protein comBiochim. Biophys. Acta, 168 (1968) 5 7 0 - 5 7 2
572
SHORT COMMUNICATIONS
TABLE llI TOTAL PEAK AREA V'/ITH INCREASING MAIN GEL CONCENTRATION
Electrophoresis was terminated in all cases when the human serum albunlin tracking dye COnlplex penetrated ~, inch into the main gel. (;el co~cn. (%)
.-trea (cm '2 × 2.5)" 4 ° l~g protein ±6o t~gprotein
3.75 5.0 7.5 15.{)
i2,3 15.o 2o.o 2 i,3
25.7 4t.o 5o.o 49.5
* Areas are the averages of 3 gels except the 16o-#g sample at J5% gel concentration which is a duplicate. pression and protein spreading, when considering quantitation of acrylamide gel patterns by the densitometer. Compression gave a nonlinear increase in the total peak area as the q u a n t i t y of protein was increased. Spreading effects are evident from the data given in Tables II and I I I . Increasing penetration into the main gel or increasing the main gel concentration to 7.5% increased the spreading of the sample. Concomitant with the increased spreading was an increase in total peak area. These data have (I) emphasized the high sensitivity of the disc technique by demonstrating t h a t protein quantities above 15 #g are beyond the normal working range of the densitometer ; (2) pointed out that, for the same sample size, peak areas are greatly influenced b y the concentration of the main gel; (3) stressed the need for migrating the protein bands to fixed distances into the main gel. When dealing with biological mixtures of proteins such as those present in serum, it is clearly not possible to preset either the sample size or the penetration distance of all the components in the mixture to their o p t i m u m range. The components present in large amounts and the components of high molecular weight t h a t do not penetrate sufficiently will appear to be present in smaller proportion in the mixture than they actually are. Thus, these results underline the difficulties in interpreting the results from the densitometer traces of acrylamide gel patterns. This work was supported b y a grant from the Chicago and Illinois Heart Associations, a grant CAO-I932-I 5 from U.S. Public Health Service and a grant from the American Cancer Society. A. W. K. is a U.S. Public Health Service trainee on grant TI-GM-653, and K. A. N. is the recipient of a research career development award 5K3-CA-3 I, o63-o3 from the National Cancer Institute. T h e B u r n s i d e s Research Laboratory, U r b a n a , Ill. 6 1 8 o z ( U . S . A . ) I 2 3 4 5
ARTHUR
W.
KRUSKI
K. ANANTH NARAYAN
1.. ()RNSTE]N, :~l'ln. N . Y . Acad. Sci., 121 (I964) 3 2 L B. J . IDAVIG, , t m z . N . Y . :4cad. Sci., I 2 l (1904) 4 0 4 . 1"2.A. NARAYAN, S. NARAYAN AND F. A. KUMMERO\V, Clin. Chim. ~4cla, 14 (i9(}0) 227, D. M. FAMBI~OUGH, F. I~'UJIMURA AND J. BONNF.I% Biochemistry, 7 (~908) 575. l<. A. NARAYAN, Lipids, 2 (1907) 282.
Received September ISth, 1968 l:tiochim, t4iophys. Acta, I(i8 (1908) 57° -572
SHORT COMMUNICATIONS
33121
Some quantitative aspects of disc
electrophoresis
Although the disc electrophoretic technique of ORNSTEIN1 and DAVIS2 has become widely accepted as a qualitative tool for the resolution of proteins, quantitative data on the separated protein components is sparse a,4. While investigating the disc electrophoretic patterns of serum lipoproteins of several species, an anomaly in the peak area of the low density lipoproteins at different gel concentrations was observed a. In order to clarify this, we have extended our investigation to some of the quantitative aspects of the disc electrophoretic method using well-characterized proteins such as egg ovalbumin and hmnan serum albumin. We report here on factors such as gel concentration, penetration distances of protein into the main gel and sample quantities, which we observed to have a profound influence on densitometer peak areas. The equipment used in this study was the Model Ie Disc Electrophoresis Unit and the Model E Microdensitometer (Canalco, Rockville, Md.). Egg ovalbunfin (5 times crystallized), human serum albumin grade I I I (Sigma Chemical Co., St. Louis, Mo.) and Amidoblack IoB (Merck AG, Darmstadt, Germany) were the proteins and dye used in this report. The disc electrophoretic procedure was the same as Method C of NARAYAN, NARAYANAND KUM~IEROWa. Volumes of 1oo #1 were loaded for all ovalbumin quantities, except for the I6o-#g samples where 2o0 ffl were used. Unless otherwise stated, the main gel was z l inch long and the spacer gel was ½ inch long. The electrophoresis was terminated when the tracking dye (Bromphenol Blue) penetrated ~ inch into the main gel, except for the penetration studies where different distances of penetration were employed. After electrophoresis, the gels were stained in a 7-5% aq. acetic acid solution containing i ~Yo Amidoblack dye. Staining was allowed to proceed for ~-2 h. Staining times up to 8 h did not affect the areas under the densitometric pattern for the same sample size. Destaining was accomplished by repeatedly washing the gels over a period of 2- 4 days with a 7.5 % aq. acetic acid solution. Electrophoretic destaining, performed immediately after the staining time, gave tile same results as the washing procedure. The entire area under the densitometric pattern was used in obtaining the peak area and will henceforth be referred to as the total peak area. The peak areas were determined by counting the number of integrator pips under each peak a. The microdensitometer, with a tungsten light source, was fitted with a Wratten 24 gelatin filter and was checked by using neutral density filters. The integrator of the densitometer was also checked for accuracy by determining the nmnber of pips per I2 inch on chart paper at different absorbance settings, ranging from o.z to 4.I, using either the control knob on tile densitometer alone or in conjunction with the neutral density filters. It was observed that the integrator response was linear up to an absorbance of 3.0. The results indicated that there was a linear relationship in the total peak area with increasing amounts of protein only up to I5 #g of protein (Table I). With as little as lO/~g of ovalbumin, the maximum absorbance observed on tile densitometer was already ~.2 and served to emphasize the great sensitivity of tile technique. Samples containing as little as I #g of protein were detected visually as well as by the densitometer. Ovalbumin resolved into one main band and 3 4 minor bands. The minor bands Biochim. Bioph),s. Acla, 168 (1968) 57° 572
571
SHORT COMMUNICATIONS TABLE
I
TOTAL PEAK AREA WITH INCREASING AMOUNTS OF OVALBUMIN The concentration
o f m a i n g e l u s e d w a s 7.5 % .
Protein* (l~g)
Area N u m b e r of (cm 2 × 2.5) ** determinations
5 io 15 20 4° 80 16o
0.75 5.1 = lO. 3 ~= 14.7 ~= I8.4 ± 26.0 i 38.5 ± 5 °.6 =
0.8 I.O 1.3 0.9 2.t 1.9 3.9
2 8 9 5 9 18 7 8
* B o t h d y e a n d p r o t e i n a r e u n c o r r e c t e d f o r m o i s t u r e c o n t e n t i n all t h e T a b l e s . T h e m o i s t u r e c o n t e n t o f t h e o v a l b u m i n a n d A m i d o b l a c k i o B d y e w a s 8.2 % a n d 9 . 4 % , r e s p e c t i v e l y . *" V a l u e s g i v e n a r e t h e m e a n -k S . D .
TABLE
II
TOTAL PEAK AREA WITH INCREASING SAMPLE PENETRATION INTO THE MAIN GEL The concentration
Penetration (inch)
o f m a i n gel u s e d w a s 7 . 5 % .
Area (cm 2 × 2.5)* 40 I~g protein I6o itg protein
~1 16
I~ Ii~6
t4"° 23"7 25.3 29-7
27"3 5° 57 68.3
* A r e a s a r e a v e r a g e s o f 3 g e l s e a c h e x c e p t f o r t h e 11~ a n d i{t i n c h v a l u e s f o r t h e i 6 o - p g samples which are single determinations.
became increasingly less visible below 4 °/~g of protein sample. The total peak area became larger as the protein was allowed to penetrate increasing distances into the main gel. The total area more than doubled for each sample quantity depending on the degree of penetration into the main gel (Table II). Because of varying degrees of sample penetration at different gel concentrations, it was necessary to migrate the protein rather than the tracking dye to a preset distance. Since ovalbumin does not bind the tracking dye, human serum albumin, which appears as a purple band behind the tracking dye, was used in these experiments (Table III). As the gel concentration was increased from 3.75 to 7.5%, the total peak area increased 65 and 95%, respectively, when 4o and 16o/~g of protein were used. Increasing the gel concentration above 7.5 % had little effect, at least in the case of the small protein, human serum albumin. With large molecular weight substances, such as low density lipoproteins of human serum (mol. wt. 3.2" IO6), increases in gel concentration beyond 3.75 % caused compression rather than spreading and therefore gave rise to lower peak areas 5. The results of this study indicated the importance of two factors, protein comBiochim. Biophys. Acta, 168 (1968) 5 7 0 - 5 7 2
572
SHORT COMMUNICATIONS
TABLE llI TOTAL PEAK AREA V'/ITH INCREASING MAIN GEL CONCENTRATION
Electrophoresis was terminated in all cases when the human serum albunlin tracking dye COnlplex penetrated ~, inch into the main gel. (;el co~cn. (%)
.-trea (cm '2 × 2.5)" 4 ° l~g protein ±6o t~gprotein
3.75 5.0 7.5 15.{)
i2,3 15.o 2o.o 2 i,3
25.7 4t.o 5o.o 49.5
* Areas are the averages of 3 gels except the 16o-#g sample at J5% gel concentration which is a duplicate. pression and protein spreading, when considering quantitation of acrylamide gel patterns by the densitometer. Compression gave a nonlinear increase in the total peak area as the q u a n t i t y of protein was increased. Spreading effects are evident from the data given in Tables II and I I I . Increasing penetration into the main gel or increasing the main gel concentration to 7.5% increased the spreading of the sample. Concomitant with the increased spreading was an increase in total peak area. These data have (I) emphasized the high sensitivity of the disc technique by demonstrating t h a t protein quantities above 15 #g are beyond the normal working range of the densitometer ; (2) pointed out that, for the same sample size, peak areas are greatly influenced b y the concentration of the main gel; (3) stressed the need for migrating the protein bands to fixed distances into the main gel. When dealing with biological mixtures of proteins such as those present in serum, it is clearly not possible to preset either the sample size or the penetration distance of all the components in the mixture to their o p t i m u m range. The components present in large amounts and the components of high molecular weight t h a t do not penetrate sufficiently will appear to be present in smaller proportion in the mixture than they actually are. Thus, these results underline the difficulties in interpreting the results from the densitometer traces of acrylamide gel patterns. This work was supported b y a grant from the Chicago and Illinois Heart Associations, a grant CAO-I932-I 5 from U.S. Public Health Service and a grant from the American Cancer Society. A. W. K. is a U.S. Public Health Service trainee on grant TI-GM-653, and K. A. N. is the recipient of a research career development award 5K3-CA-3 I, o63-o3 from the National Cancer Institute. T h e B u r n s i d e s Research Laboratory, U r b a n a , Ill. 6 1 8 o z ( U . S . A . ) I 2 3 4 5
ARTHUR
W.
KRUSKI
K. ANANTH NARAYAN
1.. ()RNSTE]N, :~l'ln. N . Y . Acad. Sci., 121 (I964) 3 2 L B. J . IDAVIG, , t m z . N . Y . :4cad. Sci., I 2 l (1904) 4 0 4 . 1"2.A. NARAYAN, S. NARAYAN AND F. A. KUMMERO\V, Clin. Chim. ~4cla, 14 (i9(}0) 227, D. M. FAMBI~OUGH, F. I~'UJIMURA AND J. BONNF.I% Biochemistry, 7 (~908) 575. l<. A. NARAYAN, Lipids, 2 (1907) 282.
Received September ISth, 1968 l:tiochim, t4iophys. Acta, I(i8 (1908) 57° -572