Electrophoresis 1992, 13, 803-804
Walter Mayer Hermann Hortner Bundesanstalt fur Lebensmitteluntersuchung und -forschung, Wien
Discontinuous electrophoresis of B-caseins
803
Discontinuous electrophoresis of P-caseins for the determination of bovine caseins in milk and dairy products Discontinuous acidic anodic polyacrylamide gel electrophoresis enables the separation of bovine @-caseinsfrom those of ovine and caprine. Interfering protein bands as a consequence of ripening or processing have not been detected. After evaluation of the stained gel by laser densitometry, quantification was performed with calibration standards on the same gel by the ratio of the peak areas from bovine to ovine and bovine to caprine, respectively. Thus, independence from the extractability of proteins affected by denaturation and ripening (which might in some cases raise the limit of detection) is achieved.The range of quantifcation extends from 5 to approximately 70% bovine casein in relation to total casein.
Various electrophoretic methods have been proposed for the detection of bovine milk in ovine and caprine milk and their dairy products [l-41. Caseins, representing the main part of the fat-free dry matter, are the preferred analytical components.They are relatively stable against heat and can be isolated easily. The @-caseinsmake up about 20 O/o of the whole casein and are much more resistant against proteolysis than a-caseins [4]. The presented acidic discontinuous polyacrylamide gel electrophoresis (PAGE) clearly separates p-caseins by selective unstacking. All chemicals were of analytical grade. Piperazine diacrylamide (PDA) was from Bio-Rad and N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) from Sigma. Caseins from milk and cheese (if necessary, suspended in water) were precipitated at pH 4.6. The precipitate was washed once with acidified water, three times with acetone, and dried. Ten mg of dry casein powder were dissolved in 5 mL of Correspondence: Dr. Hermann Hijrtner, Bundesanstalt fur Lebensmitteluntersuchung und -forschung, Kinderspitalgasse 15,A-1090Wien, Austria
Abbreviations: ACES, N-(2-acetamido)-2-am ino-ethanesulfonic acid; %C, proportion of crosslinker in T; PAGE,polyacrylamide gelelectrophoresis; PDA, piperazine diacrylamide; %T,total monomer concentration
sample buffer (containing 8 M urea, 82 mM lactic acid, 24 mM dithiothreitol (DTT), 2 4 m EDTA, ~ 8% w/v glycerol and Bromphenol Blue as marker dye; pH was brought to 4.75 with pyridine). Applied sample volume was 10 pL. Gel and buffer solutions were prepared according to [5,6] with several modifications. The resolving gel, containing 8 M urea, was 5 O/o T and 2.6 O/o C (PDA) with 50 mM lactic acid as leading ion. One hundred I.ILN,N,N,N'-tetramethylethylenediamine (TEMED)/100 mL gel solution were added and the pH was adjusted to 5.75 with pyridine. After degassing, polymerization was initiated with 137 mg potassium persulfate (KP)/100 mL gel solution. The stacking gel, containing 8 M urea, was 3.5% T and 2.6% C (PDA) with 100 mM lactic acid as leading ion and titrated to pH 4.75 with pyridine. After degassing, 24 mg AgNO, and 109 mg KP were added to 100 mLgel solution for polymerization.The upper buffer (catholyte) was 40 mM ACES (trailing ion), adjusted to pH 6.8 with KOH. The lower buffer (anolyte) - thermostated at 7" C and stirred during the run - was 200 mM pyridine (common ion) titrated to pH 5.2 with HCl. Electrophoresis was carried out at 3.5 mA/cm2 and 10 mA/cm2gel cross-section through the stacking and resolving gel, respectively. The gel was fixed and stained in a single step with 0.05 O/o w/v Coomassie Brilliant Blue G-250, dissolved in 12.5% w/v trichloroacetic acid (TCA) and destained in 2.5% w/v TCA [7].
4K2 4Kl
0VCH Verlagsgesellschaft mbI-I, D-6940 Weinhelm, 1992
Ftgure I . Separation of calibration mixtures. (a) Bovinelovine casein, from left to right: 75/25,67/33,50/50,33/67,25/75,171
83,10/90,5/95,3/97and0/100.(b)Bovine/ caprine casein, from left to right: 3/97, 5/95,10/90,17/83,25/75,33/67,50/50,67/ 33, 75/25 and 100/0. 0173-0835/92/0910-0803 $3.50+ 25/0
804
W. M a y r and H. Hortner
ElectrophorrPis 1992, f3, 803-804
Bovine p-caseins (identification not shown) appear as two major protein bands with relative mobilities of 0.82 (K,) and 0.75 (KJ. The relative mobilities of ovine p-caseins and caprine p-caseins were 0.85 and 0.78 for S, and S,, respectively, and 0.87 and 0.80 for (Z,) and (ZJ, respectively (Fig. la, Ib). Densitometryand integration of peaks were carried out on a laser-densitometer (LKB 2202 Ultroscan) connected to a PC with the appropriate software (Fig. 2). For the determination of bovine casein (5-70% in relation to total casein contents) of an unknown sample, the ratio of peak area K , to peak area S, or Z, was calculated. Calibration was done on the same gel with casein mixtures (Fig. 3). A visual detection of 2-3% bovine casein was possible.
0 0 i 0
X
m mLI m Y CI lLI 0
0 u i
a
Polymerization of acidic gel solutions is known to be difficult and modified catalytic systems are needed [6,8].
B o v i n e casein conc.
(8)
Fignre 3. Calibration for (a) bovine/ovine and (b) bovinelcaprine caseinmixtures (separations shown in Fig. l a and lb).
2
-I
II:
AgNO, instead of T E M E D for the stacking gel was effective. The use of PDA as crosslinking agent improved the separation of proteins [9] and reduced background staining when purified Coomassie Blue [7] was used. By applying low pH values for anodic discontinuous PAGE and selective unstacking of 6-caseins, we effectively separated bovine p-caseins from p-caseins of ovine and caprine. The a-caseins and proteolytic degradation products remain stacked. Although p-caseins of ovine and caprine exhibit different mobilities, sufficient discrimination in mixtures could not be achieved. Our experience with a great number of samples (commercially available as well as specially prepared for study) will be presented and discussed later.
'2
The authors wish to thank Dr. Erich Gombocz f o r developing the sophisticated software. Received August 10, 1992
References
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-
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0
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2
Gel length (rnrn) Figure 2. Laser densitometric evaluation of a separation of bovine/ovine mixture, 50/50. Bovine and ovine &caseins are labeled K1,K, and S , , S2, respectively, in order of decreasing mobi1ity.The large peak (not labeled) at 5 mm gel length represents the proteins in the stack. Only the lowerpart of the gel, containing the proteins of interest and the stack, was subjected to densitometry.
[ l ] Addeo, F., Moio, L., Chianese, L., Stingo, C., Resmini, P., Berner,I., Krause, I., Di Luccia, A. and Bocca, A., Milchwissenschaft 1990,4j, 708-711. [2] Trieu-Cuot, P. and Gripon, J.C., J. Dairy Res. 1981, 48, 303-310. [3] Ramos, M., Sanchez, R.M. and Olano, A , , Chem. Mikrobiol. Technol. Lebensm. 1985, 9,24-21. [4] Windemann, H., Tobler, M. and Baumgartner, E., Mitt. Gebiete Lebensm. Hyg. 1986, 77,565-582. [5] Chrambach, A , The Practice ofQuantitative CelElectrophoresis,VCH Verlagsgesellschaft, Weinheini 1985, pp. 1-265. [6] Maurer, H.R., Disc Electrophoresis, 2nd. Edit., W. de Gruyter, Berlin 1971, pp. 32-109. [7] iVeuhoff,V.,Stamm,R.and Eibl,H.,Electrophoresis 1985,6,427-448. [Z] Andrews, AT., Electrophoresis, 2nd. Edit., Clarendon Press, Oxford 1987, pp. 19-24. [Y] Hochstrasser, D.F. and Merrill, C.R., Appl. Theor. Electrophoresis 1988, I , 35-40.
Walter Mayer Hermann Hortner Bundesanstalt fur Lebensmitteluntersuchung und -forschung, Wien
Discontinuous electrophoresis of B-caseins
803
Discontinuous electrophoresis of P-caseins for the determination of bovine caseins in milk and dairy products Discontinuous acidic anodic polyacrylamide gel electrophoresis enables the separation of bovine @-caseinsfrom those of ovine and caprine. Interfering protein bands as a consequence of ripening or processing have not been detected. After evaluation of the stained gel by laser densitometry, quantification was performed with calibration standards on the same gel by the ratio of the peak areas from bovine to ovine and bovine to caprine, respectively. Thus, independence from the extractability of proteins affected by denaturation and ripening (which might in some cases raise the limit of detection) is achieved.The range of quantifcation extends from 5 to approximately 70% bovine casein in relation to total casein.
Various electrophoretic methods have been proposed for the detection of bovine milk in ovine and caprine milk and their dairy products [l-41. Caseins, representing the main part of the fat-free dry matter, are the preferred analytical components.They are relatively stable against heat and can be isolated easily. The @-caseinsmake up about 20 O/o of the whole casein and are much more resistant against proteolysis than a-caseins [4]. The presented acidic discontinuous polyacrylamide gel electrophoresis (PAGE) clearly separates p-caseins by selective unstacking. All chemicals were of analytical grade. Piperazine diacrylamide (PDA) was from Bio-Rad and N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) from Sigma. Caseins from milk and cheese (if necessary, suspended in water) were precipitated at pH 4.6. The precipitate was washed once with acidified water, three times with acetone, and dried. Ten mg of dry casein powder were dissolved in 5 mL of Correspondence: Dr. Hermann Hijrtner, Bundesanstalt fur Lebensmitteluntersuchung und -forschung, Kinderspitalgasse 15,A-1090Wien, Austria
Abbreviations: ACES, N-(2-acetamido)-2-am ino-ethanesulfonic acid; %C, proportion of crosslinker in T; PAGE,polyacrylamide gelelectrophoresis; PDA, piperazine diacrylamide; %T,total monomer concentration
sample buffer (containing 8 M urea, 82 mM lactic acid, 24 mM dithiothreitol (DTT), 2 4 m EDTA, ~ 8% w/v glycerol and Bromphenol Blue as marker dye; pH was brought to 4.75 with pyridine). Applied sample volume was 10 pL. Gel and buffer solutions were prepared according to [5,6] with several modifications. The resolving gel, containing 8 M urea, was 5 O/o T and 2.6 O/o C (PDA) with 50 mM lactic acid as leading ion. One hundred I.ILN,N,N,N'-tetramethylethylenediamine (TEMED)/100 mL gel solution were added and the pH was adjusted to 5.75 with pyridine. After degassing, polymerization was initiated with 137 mg potassium persulfate (KP)/100 mL gel solution. The stacking gel, containing 8 M urea, was 3.5% T and 2.6% C (PDA) with 100 mM lactic acid as leading ion and titrated to pH 4.75 with pyridine. After degassing, 24 mg AgNO, and 109 mg KP were added to 100 mLgel solution for polymerization.The upper buffer (catholyte) was 40 mM ACES (trailing ion), adjusted to pH 6.8 with KOH. The lower buffer (anolyte) - thermostated at 7" C and stirred during the run - was 200 mM pyridine (common ion) titrated to pH 5.2 with HCl. Electrophoresis was carried out at 3.5 mA/cm2 and 10 mA/cm2gel cross-section through the stacking and resolving gel, respectively. The gel was fixed and stained in a single step with 0.05 O/o w/v Coomassie Brilliant Blue G-250, dissolved in 12.5% w/v trichloroacetic acid (TCA) and destained in 2.5% w/v TCA [7].
4K2 4Kl
0VCH Verlagsgesellschaft mbI-I, D-6940 Weinhelm, 1992
Ftgure I . Separation of calibration mixtures. (a) Bovinelovine casein, from left to right: 75/25,67/33,50/50,33/67,25/75,171
83,10/90,5/95,3/97and0/100.(b)Bovine/ caprine casein, from left to right: 3/97, 5/95,10/90,17/83,25/75,33/67,50/50,67/ 33, 75/25 and 100/0. 0173-0835/92/0910-0803 $3.50+ 25/0
804
W. M a y r and H. Hortner
ElectrophorrPis 1992, f3, 803-804
Bovine p-caseins (identification not shown) appear as two major protein bands with relative mobilities of 0.82 (K,) and 0.75 (KJ. The relative mobilities of ovine p-caseins and caprine p-caseins were 0.85 and 0.78 for S, and S,, respectively, and 0.87 and 0.80 for (Z,) and (ZJ, respectively (Fig. la, Ib). Densitometryand integration of peaks were carried out on a laser-densitometer (LKB 2202 Ultroscan) connected to a PC with the appropriate software (Fig. 2). For the determination of bovine casein (5-70% in relation to total casein contents) of an unknown sample, the ratio of peak area K , to peak area S, or Z, was calculated. Calibration was done on the same gel with casein mixtures (Fig. 3). A visual detection of 2-3% bovine casein was possible.
0 0 i 0
X
m mLI m Y CI lLI 0
0 u i
a
Polymerization of acidic gel solutions is known to be difficult and modified catalytic systems are needed [6,8].
B o v i n e casein conc.
(8)
Fignre 3. Calibration for (a) bovine/ovine and (b) bovinelcaprine caseinmixtures (separations shown in Fig. l a and lb).
2
-I
II:
AgNO, instead of T E M E D for the stacking gel was effective. The use of PDA as crosslinking agent improved the separation of proteins [9] and reduced background staining when purified Coomassie Blue [7] was used. By applying low pH values for anodic discontinuous PAGE and selective unstacking of 6-caseins, we effectively separated bovine p-caseins from p-caseins of ovine and caprine. The a-caseins and proteolytic degradation products remain stacked. Although p-caseins of ovine and caprine exhibit different mobilities, sufficient discrimination in mixtures could not be achieved. Our experience with a great number of samples (commercially available as well as specially prepared for study) will be presented and discussed later.
'2
The authors wish to thank Dr. Erich Gombocz f o r developing the sophisticated software. Received August 10, 1992
References
O
-
(3
N
0
m
0
*
0
m
a
0
e
0
.
l
0
n
0
G
?
0
0
2
Gel length (rnrn) Figure 2. Laser densitometric evaluation of a separation of bovine/ovine mixture, 50/50. Bovine and ovine &caseins are labeled K1,K, and S , , S2, respectively, in order of decreasing mobi1ity.The large peak (not labeled) at 5 mm gel length represents the proteins in the stack. Only the lowerpart of the gel, containing the proteins of interest and the stack, was subjected to densitometry.
[ l ] Addeo, F., Moio, L., Chianese, L., Stingo, C., Resmini, P., Berner,I., Krause, I., Di Luccia, A. and Bocca, A., Milchwissenschaft 1990,4j, 708-711. [2] Trieu-Cuot, P. and Gripon, J.C., J. Dairy Res. 1981, 48, 303-310. [3] Ramos, M., Sanchez, R.M. and Olano, A , , Chem. Mikrobiol. Technol. Lebensm. 1985, 9,24-21. [4] Windemann, H., Tobler, M. and Baumgartner, E., Mitt. Gebiete Lebensm. Hyg. 1986, 77,565-582. [5] Chrambach, A , The Practice ofQuantitative CelElectrophoresis,VCH Verlagsgesellschaft, Weinheini 1985, pp. 1-265. [6] Maurer, H.R., Disc Electrophoresis, 2nd. Edit., W. de Gruyter, Berlin 1971, pp. 32-109. [7] iVeuhoff,V.,Stamm,R.and Eibl,H.,Electrophoresis 1985,6,427-448. [Z] Andrews, AT., Electrophoresis, 2nd. Edit., Clarendon Press, Oxford 1987, pp. 19-24. [Y] Hochstrasser, D.F. and Merrill, C.R., Appl. Theor. Electrophoresis 1988, I , 35-40.
