ANALYTICAL
65, 533-536
BIOCHEMISTRY
Recent
Staining
(1975)
Artifacts
on Gel
with
LKB “Ampholines”
Isoelectric-Focusing
WENDY I. OTAVSKY AND JAMES W. DRYSDALE' Department
of Biochemistry and Pharmacology. 136 Harrison Alvnue. Boston, Received
November
18. 1974:
Tufts Urziversity School Mmsaclursetts 021 I I accepted
January
of Medicine.
8, 1975
Certain batches of LKB “Ampholines” gave multiple bands when stained after gel isoelectric-focusing with commonly used direct staining procedures. The stainable species are most prominent in the pH range 6-9. By contrast, ampholytes prepared in the laboratory gave no stainable species in this pH range.
Isoelectric focusing is an equilibrium method for segregating amphoteric macromolecules such as proteins according to their isoelectric points (1). Separations are achieved by banding samples in stable pH gradients established by electrolysis of carrier ampholytes in an anticonvective medium such as a sucrose density gradient. Several adaptations of the original method have recently been described (2,3). Among the more promising is the use of gels of polyacrylamide or Sephadex (4) which offer resolution superior to that obtainable in sucrose density gradients. At present there is only one commercial source of carrier ampholytes (“Ampholines”, LKB Produkter AB, Bromma, Sweden) suitable for isoelectric focusing. These preparations form smooth and stable pH gradients between pH 3 and 10 and have proven adequate for many purposes. Unfortunately, however, they interfere with many methods for detecting proteins. Particularly troublesome, in analytical electrofocusing, is their reactivity with many protein stains (3). This problem was initially overcome by fixing focused proteins in acid and eluting the ampholytes by exhaustive washing, a cumbersome and time-consuming process. Recently, however, several successful methods have been described for direct staining of proteins after gel electrofocusing (3,5,6). We would like to point out that such methods may not always be applicable for all Ampholine pH ranges, possibly because of variations in recent batches. By contrast, ampholytes prepared according to the original LKB patent (7) gave no background staining. ’ To whom
all correspondence
should
be addressed. 533
Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
534
OTAVSKY
AND
MATERIALS
DRYSDALE
AND
METHODS
“Ampholines” in the following pH ranges were purchased from LKB Produkter AB, Bromma, Sweden: 3-l 0 (Lot I6 from 3-74); 4-6 (Lot 9 from 8-74); and 6-8 (Lot 3 from 5-73). Ampholyte preparation D-5, prepared according to the method of Vinogradov rt al. (7), was the generous gift of Dr. Joseph C. Bagshaw, Wayne State University, Detroit, MI. Acrylamide, N, N’-methylene-his-acrylamide (bis), both recrystallized, TEMED and Coomassie Brilliant Blue R-250 were purchased from Bio-Rad Laboratories. All other reagents were the highest grade available. Gel electrofocusing in cylinders of polyacrylamide gel was performed in apparatus from MRA Corporation (Boston, MA) as described previously (8). Gels were made of acrylamide with methylene-bisacrylamide as cross linker with the composition T = 4%, C = 4%, where T = g acrylamide + g bis per 100 ml and C = (g bis per 100 ml)/T. Focused gels were stained overnight in a solution containing 0.5% CuSO,, 0.05% Coomassie Brilliant Blue, 10% acetic acid and 37% ethanol (3). They were then removed and placed in a solution of 0.0 I % Coomassie Brilliant Blue in IO% acetic acid and 25% ethanol for approximately 6 hr. The gels were destained in a solution of 10% acetic acid and 10% ethanol and stored in 10% acetic acid. RESULTS AND
DISCUSSION
Table 1 shows the pH gradients established by the various ampholyte preparations. LKB and D-5 ampholytes established pH gradients after TABLE OF AMPHOLYTE
PH GRADIENTS LKB
1 SOLUTIONS
ON GELS"
Ampholines
3-10
6-8
4-6
D-5
4.0 4.3 4.7 5.2 5.8 6.4 7.0 7.9 8.5 9.0
4.4 5.8 6.0 6.3 6.5 6.8 7.0 7.2 1.6
4.1 4.2 4.4 4.5 4.5 4.6 4.8 4.9 5.1
3.9 4.2 4.4 4.8 5.2 5.8 6.6 1.4 8.2 8.8
a pH measurements of l-cm sections lytes were eluted from l-cm segments at 20°C.
of focused in distilled
gels of the various preparations. water and the pH measured
Ampho1 hr later
STAINING
ARTIFACTS
FIG. 1. Stained and destained focused 6-8: C, 4-6: D, D-5. A. B, and C were
WITH
535
“AMPHOLINES”
gels using the following ampholytes: produced with LKB Ampholines.
A. 3-10:
B.
about 3 hr that remained relatively stable over a further 18 hr period. Figure 1 shows that there are stainable species in LKB Ampholines pH 3-10 and 6-8 but not in the pH range 4-6. From a comparison of the stained gels with the pH gradients, it is apparent that most of the stained ampholytes in the LKB preparations have a pH above 6.5. Although the pH gradient from the D-5 ampholytes also extended to pH 9, the destained gels were perfectly clear. Figure 2 shows the confusion that arises from background staining of LKB ampholytes. Without complicated scanning procedures, direct densitometric quantitation of the focused proteins is obviously not feasible. The staining method used in these studies is similar to others developed for direct staining and has in the past given no background staining from LKB Ampholines. The staining material did not represent contamination or polymerization of ampholytes on prolonged storage since it was present in recent batches of freshly opened bottles of Ampholines. The present problems may, therefore, be due to variation in recent
FIG.
same
2. Focused stained gels using pH range with focused proteins.
LKB
Ampholines
pH
range
3-10.
A. Blank
gel:
B,
536
OTAVSKY
AND
DRYSDALE
batches or to the presence of new components that may have been added to bolster the notoriously weak pH 5-8 range (3). Unfortunately, the exact composition or batch variability of Ampholines still remains unknown. Whatever the explanation, it is clearly advisable when using LKB Ampholines to stain a blank gel to avoid problems in interpretation of stained patterns. The fact that ampholytes prepared in the laboratory do not stain under similar conditions enhances their attractive low cost and simple preparation. ACKNOWLEDGMENT This Health.
work
was
supported
by Grant
No.
AM
17775
from
the
National
Institutes
of
REFERENCES 1. 2. 3. 4. 5. 6.
Vesterberg. O., and Svensson. H. (1966) Acra Chem. Scnnd. 20, 820-834. Catsimpoolas. N. ( 1973) Sc~par. Sc,i. 8, 7 1- I2 1. Righetti, P. G.. and Drysdale, J. W. (1974) J. Chvonufogr. 98, 27 l-33 1. Radola. B. J. (1973) Ann. N. Y. Acnd. Sri. 209, 127-143. Stiderholm, J., Allestam. P.. and Wadstrem, T. (1972) FEBS Len. 24, 89-92. Vesterberg. 0. (1971) in Methods in Enzymology (Jacoby, W. B.. ed.) Vol. pp. 389-412. Academic Press, New York. 7. Vindogradov. S. N., Lowenkron, S., Andonian. H. R., Bagshaw, J.. Felgenhauer, and Pak, S. J. (1973) Biochem. Biophys. Res. Commun. 54, 501-506. 8. Righetti, P. G., and Drysdale, J. W. ( 1971) Biochim. Biophys. Acta 236, 17-28.
22. K.,
65, 533-536
BIOCHEMISTRY
Recent
Staining
(1975)
Artifacts
on Gel
with
LKB “Ampholines”
Isoelectric-Focusing
WENDY I. OTAVSKY AND JAMES W. DRYSDALE' Department
of Biochemistry and Pharmacology. 136 Harrison Alvnue. Boston, Received
November
18. 1974:
Tufts Urziversity School Mmsaclursetts 021 I I accepted
January
of Medicine.
8, 1975
Certain batches of LKB “Ampholines” gave multiple bands when stained after gel isoelectric-focusing with commonly used direct staining procedures. The stainable species are most prominent in the pH range 6-9. By contrast, ampholytes prepared in the laboratory gave no stainable species in this pH range.
Isoelectric focusing is an equilibrium method for segregating amphoteric macromolecules such as proteins according to their isoelectric points (1). Separations are achieved by banding samples in stable pH gradients established by electrolysis of carrier ampholytes in an anticonvective medium such as a sucrose density gradient. Several adaptations of the original method have recently been described (2,3). Among the more promising is the use of gels of polyacrylamide or Sephadex (4) which offer resolution superior to that obtainable in sucrose density gradients. At present there is only one commercial source of carrier ampholytes (“Ampholines”, LKB Produkter AB, Bromma, Sweden) suitable for isoelectric focusing. These preparations form smooth and stable pH gradients between pH 3 and 10 and have proven adequate for many purposes. Unfortunately, however, they interfere with many methods for detecting proteins. Particularly troublesome, in analytical electrofocusing, is their reactivity with many protein stains (3). This problem was initially overcome by fixing focused proteins in acid and eluting the ampholytes by exhaustive washing, a cumbersome and time-consuming process. Recently, however, several successful methods have been described for direct staining of proteins after gel electrofocusing (3,5,6). We would like to point out that such methods may not always be applicable for all Ampholine pH ranges, possibly because of variations in recent batches. By contrast, ampholytes prepared according to the original LKB patent (7) gave no background staining. ’ To whom
all correspondence
should
be addressed. 533
Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
534
OTAVSKY
AND
MATERIALS
DRYSDALE
AND
METHODS
“Ampholines” in the following pH ranges were purchased from LKB Produkter AB, Bromma, Sweden: 3-l 0 (Lot I6 from 3-74); 4-6 (Lot 9 from 8-74); and 6-8 (Lot 3 from 5-73). Ampholyte preparation D-5, prepared according to the method of Vinogradov rt al. (7), was the generous gift of Dr. Joseph C. Bagshaw, Wayne State University, Detroit, MI. Acrylamide, N, N’-methylene-his-acrylamide (bis), both recrystallized, TEMED and Coomassie Brilliant Blue R-250 were purchased from Bio-Rad Laboratories. All other reagents were the highest grade available. Gel electrofocusing in cylinders of polyacrylamide gel was performed in apparatus from MRA Corporation (Boston, MA) as described previously (8). Gels were made of acrylamide with methylene-bisacrylamide as cross linker with the composition T = 4%, C = 4%, where T = g acrylamide + g bis per 100 ml and C = (g bis per 100 ml)/T. Focused gels were stained overnight in a solution containing 0.5% CuSO,, 0.05% Coomassie Brilliant Blue, 10% acetic acid and 37% ethanol (3). They were then removed and placed in a solution of 0.0 I % Coomassie Brilliant Blue in IO% acetic acid and 25% ethanol for approximately 6 hr. The gels were destained in a solution of 10% acetic acid and 10% ethanol and stored in 10% acetic acid. RESULTS AND
DISCUSSION
Table 1 shows the pH gradients established by the various ampholyte preparations. LKB and D-5 ampholytes established pH gradients after TABLE OF AMPHOLYTE
PH GRADIENTS LKB
1 SOLUTIONS
ON GELS"
Ampholines
3-10
6-8
4-6
D-5
4.0 4.3 4.7 5.2 5.8 6.4 7.0 7.9 8.5 9.0
4.4 5.8 6.0 6.3 6.5 6.8 7.0 7.2 1.6
4.1 4.2 4.4 4.5 4.5 4.6 4.8 4.9 5.1
3.9 4.2 4.4 4.8 5.2 5.8 6.6 1.4 8.2 8.8
a pH measurements of l-cm sections lytes were eluted from l-cm segments at 20°C.
of focused in distilled
gels of the various preparations. water and the pH measured
Ampho1 hr later
STAINING
ARTIFACTS
FIG. 1. Stained and destained focused 6-8: C, 4-6: D, D-5. A. B, and C were
WITH
535
“AMPHOLINES”
gels using the following ampholytes: produced with LKB Ampholines.
A. 3-10:
B.
about 3 hr that remained relatively stable over a further 18 hr period. Figure 1 shows that there are stainable species in LKB Ampholines pH 3-10 and 6-8 but not in the pH range 4-6. From a comparison of the stained gels with the pH gradients, it is apparent that most of the stained ampholytes in the LKB preparations have a pH above 6.5. Although the pH gradient from the D-5 ampholytes also extended to pH 9, the destained gels were perfectly clear. Figure 2 shows the confusion that arises from background staining of LKB ampholytes. Without complicated scanning procedures, direct densitometric quantitation of the focused proteins is obviously not feasible. The staining method used in these studies is similar to others developed for direct staining and has in the past given no background staining from LKB Ampholines. The staining material did not represent contamination or polymerization of ampholytes on prolonged storage since it was present in recent batches of freshly opened bottles of Ampholines. The present problems may, therefore, be due to variation in recent
FIG.
same
2. Focused stained gels using pH range with focused proteins.
LKB
Ampholines
pH
range
3-10.
A. Blank
gel:
B,
536
OTAVSKY
AND
DRYSDALE
batches or to the presence of new components that may have been added to bolster the notoriously weak pH 5-8 range (3). Unfortunately, the exact composition or batch variability of Ampholines still remains unknown. Whatever the explanation, it is clearly advisable when using LKB Ampholines to stain a blank gel to avoid problems in interpretation of stained patterns. The fact that ampholytes prepared in the laboratory do not stain under similar conditions enhances their attractive low cost and simple preparation. ACKNOWLEDGMENT This Health.
work
was
supported
by Grant
No.
AM
17775
from
the
National
Institutes
of
REFERENCES 1. 2. 3. 4. 5. 6.
Vesterberg. O., and Svensson. H. (1966) Acra Chem. Scnnd. 20, 820-834. Catsimpoolas. N. ( 1973) Sc~par. Sc,i. 8, 7 1- I2 1. Righetti, P. G.. and Drysdale, J. W. (1974) J. Chvonufogr. 98, 27 l-33 1. Radola. B. J. (1973) Ann. N. Y. Acnd. Sri. 209, 127-143. Stiderholm, J., Allestam. P.. and Wadstrem, T. (1972) FEBS Len. 24, 89-92. Vesterberg. 0. (1971) in Methods in Enzymology (Jacoby, W. B.. ed.) Vol. pp. 389-412. Academic Press, New York. 7. Vindogradov. S. N., Lowenkron, S., Andonian. H. R., Bagshaw, J.. Felgenhauer, and Pak, S. J. (1973) Biochem. Biophys. Res. Commun. 54, 501-506. 8. Righetti, P. G., and Drysdale, J. W. ( 1971) Biochim. Biophys. Acta 236, 17-28.
22. K.,
