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A simple microtechnique for isoelectric focusing on a density gradient
ANALYTICAL
67, 679483
BIOCHEMISTRY
A Simple
(1975)
Microtechnique
Focusing
for
on a Density
Isoelectric
Gradient
Isoelectric focusing in a density gradient can be performed on chromatographic columns of IO-15ml volume. A polyacrylamide salt bridge was used to make electrical contact between the density gradient tube and the electrode compartment. An appropriate gradient mixer can easily be made from two 1O-ml syringes. The small column permits a resolution of isoenzymes which is comparable to the separation with a commercial 1 lo-ml column. With the described apparatus one uses ten times less Ampholine and biological sample. The time of isoelectric focusing is reduced three to four times.
Isoelectric focusing in a density gradient is a preparative technique by which proteins are separated according to their apparent isoelectric points (1). Until recently the technique was used on rather large columns of 110 or 440 ml. This required expensive equipment and Ampholine, a long focusing time and a large protein sample. The need for a smaller column is apparent, especially when only a small amount of material is available. Two such microcolumns have been described (2) (3). In the present communication a different technique is described for isoelectric focusing on a microscale using apparatus made from commercially available parts. MATERIALS
AND
METHODS
A. The Gradient Mixer
A very simple gradient mixer for small volumes can be made from two plastic lo-ml syringes (Fig. 1). At the bottom of one syringe a hole (C) is made with a hot needle. This hole is connected to a peristaltic pump. A silicone rubber tube (o.d. = 4 mm, length = km) connects outlet D from the first syringe with the outlet from the second syringe A. This syringe contains 5 ml of the dense solution (32% sucrose-2% Ampholine), while chamber B contains 5 ml of the light solution (8% sucrose-2% Ampholine). When the syringes are being filled, the tube between A and D has to be tightly depressed with two fingers. A magnetic stirring bar of IO-mm length in chamber B is moved by a stirring motor, placed about 10 cm under chamber B. 679 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form resewed.
680
SHORT
COMMUNICATIONS
FIG. 1. Simple gradient mixer. A. Dense solution compartment; B, light solution compartment; C, extra hole made by insertion of a hot needle; D, magnetic stirring bar (length 10 mm).
B. The Apparatus (Fig. 2) 1. The column. Any chromatographic column with a length of 30 cm and with a diameter of 10 mm can be used. A water jacket is necessary. Cooling was achieved by a mixture of water and ethylene glycol, circulated at 4°C from a kryostat. 2. The central glass tube (Fig. 2B). The critical design of the central tube, through which the lower electrode gases can escape, was essential for the successful operation of the column. The lower electrode compartment was made of a glass tube (outer diameter, 6 mm) a few centimeters longer than the column. This central tube is filled with the lower electrolyte solution. This fluid may not be allowed to drain from the central tube at the end of the experiment. Therefore a “stopper” of polyacrylamide gel is made in situ at the bottom of the central tube. This is done in the following way: The central tube is put in a test tube. One millimeter of the polymerization mixture (containing 200 mg of acrylamide, 5 mg of NN’-methylenebisacrylamide, 5 ~1 of N, N, N’, N’-tetramethylethylenediamine and 2 mg of ammonium persulfate) is poured into this tube, leaving about 1 cm of the lower end of the central tube in
SHORT
681
COMMUNICATIONS
FIG. 2. Isoelectric focusing on a small column. A. Chromatographic tube; C. “stopper” of polyacrylamide gel; D. electrodes: E, water piece of silicone rubber tubing.
column: B, central jacket; F, clamp on a
the polymerizing mixture. After about 20 min, the glass tube can be removed and placed into the column. 3. The electrodes. Two platinum wires were used as electrodes. The lower electrode was put in the central tube, while the upper electrode was placed at the upper end of the column (Fig. 2). C. Conditions
of a Representative
Focusing
Experiment
The protein sample was mixed with the contents of one of the chambers of the gradient mixer. The column was filled about 0.2-0.4 ml/min. The “gradient solution” was pumped through the outlet tube (Fig. 2F), starting with the liquid from the light solution chamber (Fig. 1B). After the total volume of the density gradient solution was pumped into the column, it was followed by 0.5 ml of a 32% sucrose solution containing 0.1 M NaOH. Care had to be taken to exclude air bubbles in the peristaltic pump tube. To the top of the column 0.5 ml of 0.1 M NH,SO, was manually pipetted and layered above the sucrose gradient. Voltage was then applied, starting with 200 V and 1 mA. After about 16 hr the current was reduced to almost zero at 400 V. Proteins were then presumed to be focused. After termination of the focusing, the column may be drained at a rate of 20 drops/min.
682
SHORT COMMUNICATIONS
FIG. 3. Separation of acid hydrolases from human liver: N-acetyl-fi-glucosaminidase: cymannosidase; and P-galactosidase. Enzyme activity is expressed in nanomoles of substrate hydrolysed after 30 min by 20 ~1 of each fraction. (A), Isoelectric focusing in an LKB 1 lo-ml column. Sample: 500 ~1 of a liver homogenate (lo%, v/v), 3.2 mg of protein; time of focusing: 60 hr; fractions: 3ml; gradient: pH 3-10. (B), Isoelectric focusing in a Pharmacia K9-30 column with water jacket. Sample: 50 ~1 of a liver homogenate (10%. v/v) 0.31 mg protein: time of focusing: 16 hr: fractions: seven drops (about 250 ~1); gradient: pH 3-10.
D. The Collection of the Ftactions A Microcol TDC 80 fraction collector (Gilson Medical Electronics, Villiers-le-Bel, France) was used for collecting small fractions of seven drops each (about 0.35 ml). E. The Analysis A 0.25-ml sample contains enough liquid to permit a pH measurement. With a glass electrode (Type GK 2 321, Radiometer, Copen-
SHORT
6X3
COMMUNICATIONS
hagen) reproducible pH measurements were measured, using microadaptations methyl umbelliferylderivatives (4). RESULTS
AND
were obtained. of fluorimetric
Acid hydrolases procedures with
DISCUSSION
With the device described above, a linear pH gradient is obtained after about 16 hr, i.e., overnight. It is thus possible to perform an isoelectric focusing experiment every day. In Fig. 3A results of a representative experiment are shown. The data are very similar when compared with the separation obtained with an LKB IOO-ml column (Fig. 3B) after 48 hr of isoelectric focusing. The advantages of the micromethod are obvious: I), Less time is needed, which permits experiments with labile enzymes. 2), Less Ampholine is used. In a 1 lo-ml column one needs 3-5 ml of the expensive Ampholine; in the microcolumn, 0.2-0.4 ml is sufficient. 3), Less sample is needed; this may be the most important advantage. With as little as 50 pug of liver protein or with only 20 ~1 of blood plasma it was possible to analyze five hydrolases (not shown). 4), The whole apparatus is inexpensive and easy to build. CONCLUSION
Isoelectric focusing in a density gradient is possible in a volume of 10 ml. The use of a central glass tube with a “stopper” of polyacrylamide gel permits easy elution. An appropriate small density gradient mixer can be made from two disposable syringes. With the described device one used ten times less Ampholine, ten times less of the biological sample and the time for isoelectric focusing is reduced three to four times. ACKNOWLEDGMENTS I thank
Mrs.
Marleen
Van
Den
Berghe
and Mrs.
Odette
Coessens
for
skillful
help,
REFERENCES I. 2. 3. 4.
Vesterberg, 0.. and Svensson, H. (1966) Act{? Chrnz. Stand. 20, 8X-834. Osterman, L.. (1970) Sci. Tools 17, 31-33. Korant, B. D., and Lonberg, K. ( 1974) Anctl. Bioc/zem. 59, 75-82. Kint, J. A. (1973) FEES Lett. 36, 53-56.
J. A. Kliniek VOOY Kinderziekfrn Akademisch Ziekenhuis Ghent. Belgium Received June I I, 1974;
accepted
March
18, 1975
KINT
67, 679483
BIOCHEMISTRY
A Simple
(1975)
Microtechnique
Focusing
for
on a Density
Isoelectric
Gradient
Isoelectric focusing in a density gradient can be performed on chromatographic columns of IO-15ml volume. A polyacrylamide salt bridge was used to make electrical contact between the density gradient tube and the electrode compartment. An appropriate gradient mixer can easily be made from two 1O-ml syringes. The small column permits a resolution of isoenzymes which is comparable to the separation with a commercial 1 lo-ml column. With the described apparatus one uses ten times less Ampholine and biological sample. The time of isoelectric focusing is reduced three to four times.
Isoelectric focusing in a density gradient is a preparative technique by which proteins are separated according to their apparent isoelectric points (1). Until recently the technique was used on rather large columns of 110 or 440 ml. This required expensive equipment and Ampholine, a long focusing time and a large protein sample. The need for a smaller column is apparent, especially when only a small amount of material is available. Two such microcolumns have been described (2) (3). In the present communication a different technique is described for isoelectric focusing on a microscale using apparatus made from commercially available parts. MATERIALS
AND
METHODS
A. The Gradient Mixer
A very simple gradient mixer for small volumes can be made from two plastic lo-ml syringes (Fig. 1). At the bottom of one syringe a hole (C) is made with a hot needle. This hole is connected to a peristaltic pump. A silicone rubber tube (o.d. = 4 mm, length = km) connects outlet D from the first syringe with the outlet from the second syringe A. This syringe contains 5 ml of the dense solution (32% sucrose-2% Ampholine), while chamber B contains 5 ml of the light solution (8% sucrose-2% Ampholine). When the syringes are being filled, the tube between A and D has to be tightly depressed with two fingers. A magnetic stirring bar of IO-mm length in chamber B is moved by a stirring motor, placed about 10 cm under chamber B. 679 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form resewed.
680
SHORT
COMMUNICATIONS
FIG. 1. Simple gradient mixer. A. Dense solution compartment; B, light solution compartment; C, extra hole made by insertion of a hot needle; D, magnetic stirring bar (length 10 mm).
B. The Apparatus (Fig. 2) 1. The column. Any chromatographic column with a length of 30 cm and with a diameter of 10 mm can be used. A water jacket is necessary. Cooling was achieved by a mixture of water and ethylene glycol, circulated at 4°C from a kryostat. 2. The central glass tube (Fig. 2B). The critical design of the central tube, through which the lower electrode gases can escape, was essential for the successful operation of the column. The lower electrode compartment was made of a glass tube (outer diameter, 6 mm) a few centimeters longer than the column. This central tube is filled with the lower electrolyte solution. This fluid may not be allowed to drain from the central tube at the end of the experiment. Therefore a “stopper” of polyacrylamide gel is made in situ at the bottom of the central tube. This is done in the following way: The central tube is put in a test tube. One millimeter of the polymerization mixture (containing 200 mg of acrylamide, 5 mg of NN’-methylenebisacrylamide, 5 ~1 of N, N, N’, N’-tetramethylethylenediamine and 2 mg of ammonium persulfate) is poured into this tube, leaving about 1 cm of the lower end of the central tube in
SHORT
681
COMMUNICATIONS
FIG. 2. Isoelectric focusing on a small column. A. Chromatographic tube; C. “stopper” of polyacrylamide gel; D. electrodes: E, water piece of silicone rubber tubing.
column: B, central jacket; F, clamp on a
the polymerizing mixture. After about 20 min, the glass tube can be removed and placed into the column. 3. The electrodes. Two platinum wires were used as electrodes. The lower electrode was put in the central tube, while the upper electrode was placed at the upper end of the column (Fig. 2). C. Conditions
of a Representative
Focusing
Experiment
The protein sample was mixed with the contents of one of the chambers of the gradient mixer. The column was filled about 0.2-0.4 ml/min. The “gradient solution” was pumped through the outlet tube (Fig. 2F), starting with the liquid from the light solution chamber (Fig. 1B). After the total volume of the density gradient solution was pumped into the column, it was followed by 0.5 ml of a 32% sucrose solution containing 0.1 M NaOH. Care had to be taken to exclude air bubbles in the peristaltic pump tube. To the top of the column 0.5 ml of 0.1 M NH,SO, was manually pipetted and layered above the sucrose gradient. Voltage was then applied, starting with 200 V and 1 mA. After about 16 hr the current was reduced to almost zero at 400 V. Proteins were then presumed to be focused. After termination of the focusing, the column may be drained at a rate of 20 drops/min.
682
SHORT COMMUNICATIONS
FIG. 3. Separation of acid hydrolases from human liver: N-acetyl-fi-glucosaminidase: cymannosidase; and P-galactosidase. Enzyme activity is expressed in nanomoles of substrate hydrolysed after 30 min by 20 ~1 of each fraction. (A), Isoelectric focusing in an LKB 1 lo-ml column. Sample: 500 ~1 of a liver homogenate (lo%, v/v), 3.2 mg of protein; time of focusing: 60 hr; fractions: 3ml; gradient: pH 3-10. (B), Isoelectric focusing in a Pharmacia K9-30 column with water jacket. Sample: 50 ~1 of a liver homogenate (10%. v/v) 0.31 mg protein: time of focusing: 16 hr: fractions: seven drops (about 250 ~1); gradient: pH 3-10.
D. The Collection of the Ftactions A Microcol TDC 80 fraction collector (Gilson Medical Electronics, Villiers-le-Bel, France) was used for collecting small fractions of seven drops each (about 0.35 ml). E. The Analysis A 0.25-ml sample contains enough liquid to permit a pH measurement. With a glass electrode (Type GK 2 321, Radiometer, Copen-
SHORT
6X3
COMMUNICATIONS
hagen) reproducible pH measurements were measured, using microadaptations methyl umbelliferylderivatives (4). RESULTS
AND
were obtained. of fluorimetric
Acid hydrolases procedures with
DISCUSSION
With the device described above, a linear pH gradient is obtained after about 16 hr, i.e., overnight. It is thus possible to perform an isoelectric focusing experiment every day. In Fig. 3A results of a representative experiment are shown. The data are very similar when compared with the separation obtained with an LKB IOO-ml column (Fig. 3B) after 48 hr of isoelectric focusing. The advantages of the micromethod are obvious: I), Less time is needed, which permits experiments with labile enzymes. 2), Less Ampholine is used. In a 1 lo-ml column one needs 3-5 ml of the expensive Ampholine; in the microcolumn, 0.2-0.4 ml is sufficient. 3), Less sample is needed; this may be the most important advantage. With as little as 50 pug of liver protein or with only 20 ~1 of blood plasma it was possible to analyze five hydrolases (not shown). 4), The whole apparatus is inexpensive and easy to build. CONCLUSION
Isoelectric focusing in a density gradient is possible in a volume of 10 ml. The use of a central glass tube with a “stopper” of polyacrylamide gel permits easy elution. An appropriate small density gradient mixer can be made from two disposable syringes. With the described device one used ten times less Ampholine, ten times less of the biological sample and the time for isoelectric focusing is reduced three to four times. ACKNOWLEDGMENTS I thank
Mrs.
Marleen
Van
Den
Berghe
and Mrs.
Odette
Coessens
for
skillful
help,
REFERENCES I. 2. 3. 4.
Vesterberg, 0.. and Svensson, H. (1966) Act{? Chrnz. Stand. 20, 8X-834. Osterman, L.. (1970) Sci. Tools 17, 31-33. Korant, B. D., and Lonberg, K. ( 1974) Anctl. Bioc/zem. 59, 75-82. Kint, J. A. (1973) FEES Lett. 36, 53-56.
J. A. Kliniek VOOY Kinderziekfrn Akademisch Ziekenhuis Ghent. Belgium Received June I I, 1974;
accepted
March
18, 1975
KINT