378
Electrophoresis 1991, 12, 378-380
D. Megow and 0.Jacobasch
A miniaturized electrode configuration for isoelectric focusing
Dirk Megow Gisela Jacobasch Institute of Biochemistry, Humboldt-University Berlin
An electrode configuration is described which allows fast isoelectric focusing (IEF) with conventional IEF systems. The equipment, which can be fixed on the cooling plate of a conventional IEF system, consists ofa base plate on which ffappable electrode holders are fastened. The handling is simple and needs only little time. Graphite rods are used as electrodes, thus avoiding the use of buffer strips Samples are applied with special applicator strips -permitting the analysis of up to 19 samples on a 50 X 40 mm polyacrylamide gel and up to 44 samples on a 100 X 70 mm gel. Only 30 min are needed for one IEF run.
The commercially available PhastSystem (Pharmacia), which permits short separation times, differs from conventional isoelectric focusing (IEF) units mainly by the simple handling of the IEF gels. It has a new electrode configuration, in which platinized titanium rods are placed directly on the gel surface, thereby avoiding the use of electrode strips. Samples are applied automaticallywith sample applicators [I]. We present an electrode configuration which makes it possible to carry out fast IEF on conventional horizontal IEF systems. Our electrode Configuration consists of
a base plate on which flappable electrode holders are fastened by sticking strips. The base plate can be fixed on the cooling plate of a conventional IEF system by adding a small amount of distilled water into the interspace. Two variants of the electrode configuration were used: IEF was carried out either with a two-electrode system (Fig. 1A) on 50 X 40 mm polyacrylamide gels or with a three-electrode system on 100 X 70 mm gels. The configuration with three electrodes consists of one central anode and two cathodes (Fig. 1B) as introduced by Altland [2]. We used graphite rods with a diameter of 3-5 mm as electrodes and placed them directly on the IEF gels, thus avoiding the use of buffer strips.
Y
/
Figure 2. Sample applicator: Small rubber squares are attached to a rigid support. Sample volumes of 0.5-1 pL are pipetted directly onto the squares, then for sample application the applicator is placed with the squares on the IEF gel.
2500 -
v
- 2.5
i
p
Figure I . Electrode equipment: Base plate with flappable electrode holders (fixed on the base plate by adhesive strips) and graphite electrodes: (A) Conventional configuration with electrodes and (B) three-electrode system with one central anode and two cathodes. (1) Flappable electrode holders with graphite electrodes, (2) polyacrylamide gel, (3) base plate.
~~
~~~
~
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
i
/ O 5
10
15
20
25
30
rnin
~~~~
Abbreviations: IEF, isoelectric focusing, PK, pyruvate kinase
mA,W
10.5
01 0
Correspondence: Dr. Dirk Megow, Institut fur Biochemie, Humboldt-Universitat, Wessische Str. 3-4, DO-1040 Berlin, Germany
I
Figure 3. Running conditions during IEF with a 50 X 40 mm polyacryl= 2500V, I,,, = 2 mA, amide gel, 3.5% Ampholine 3.5-10: V,,,
P,,
=2
w.
0173-0835/91/0505-0378 $3.50+.25/0
Elecfropkorcsrs 1991, 12, 378-380
A miniaturized electrode configuration for isoelectric focusing
379
PH 9-
7
-
7
3
I
0
I
I
10 20 30 gel length (mm)
Figure 4. pH gradient after IEF, determined with a pH marker mixture from Pharmacia. Running conditions as shown in Fig. 3. The corresponding IEF pattern is shown in Fig. 5A (lane 9).
Figure 5. IEF runs with 50 X 40 mm polyacrylamide gels. (A) 11samples and (B) 19 samples, respectively, were applied with the applicator strips shown in Fig. 2. (A) I500 ng (11, (2); 375 ng (31, (lo), (11); 94 ng (4); 31 ng (5); 7.5 ng (6); and 2 ng (7) of hemoglobin were applied. Detection limit was 31 ng with Coomassie Brilliant Blue staining. Pharmacia marker mixture (8) stored frozen at -18OC or (9) freshly dissolved, were applied at a loading of 100 ng perprotein. (B) Alternating application of hemoglobin (62 ng) and pH marker proteins (100 ng per each protein). Seventeen samples were applied, 2 lanes were left free. A mixing of the samples or a leaking into the blank lanes was not observed using the applicator strips.
IEFwas performed with an LKB2117Multiphorseparation unit and LKB 2197 power supply (Bromma, Sweden) on 200 pm thick polyacrylamide gels (5 O/oT, 4%C) with 3.5 O/o Ampholine 5-7 or 3.5-10 (LKB) and 12% glycerol on glass plates silanized with Silane A-174. Sample application was performed with special applicator strips where small rubber pieces are attached to a rigid support (Fig. 2). Sample volumes of 0.5-1 pL were pipetted directly onto the squares and then the applicator was placed with the side of the squares on the IEF gel. Under the chosen running conditions (Fig. 3) IEF was performed in about 30 min, comparable to run times obtained with the PhastSystem [3]. No excessive heat development of the graphite rods was observed. The pH gradient (Fig. 4), as estimated by a Pharmacia pH marker mixture, was nearly linear and not distorted by secondary electrode reactions of contaminations (of the graphite material) or reactions with the carrier ampholytes. However, some oxidative corrosion of the anodic graphite material was observed after repeated
use. This corrosion effect could be diminished by using especially pure graphite charges for electrodes. Nevertheless, an interchange of cathode and anode should be avoided because some electrode material can penetrate from the corroded electrode into the gel (not shown). Sample application was fast and easy using our applicator. Up to 19 samples could be analyzed on one 50 X 40 mm polyacrylamide gel (Fig. 5B). A mixing of the alternately applied hemoglobin and pH marker samples was not observed. No leaking of any protein into the blank lanes was detected. The detection limit for proteins was about 30 ng on staining with Coomassie Brilliant Blue (Fig. 5A). Other visualization procedures could also be applied and, as an example, Fig. 6 demonstrates the microheterogeneity of human erythrocyte pyruvate kinase (PK). PK was visualized by an indirect immunoassay with diaminobenzidine staining after immunofixation [4,51. IEF was carried out with the three electrode configuration where up to 44 samples can be applied on a 100 X 70 mm gel.
380
Electrophoresis 1991, 12, 378-380
D. Megow and G. Jacobasch
Figure 6. I E F of PK on a 100 X 70 mm polyacrylamide gel using the three-electrode system (Fig. 1B). PK was visualized by diaminobenzidine staining after immunofixation with a monospecific anti-PK serum and incubation with peroxidase-labeled second antibodies. Up to 44 samples can be analyzed with one gel. Differences in sharpness and intensity between the lanes are due to sample treatment with oxidative agents, partly resulting in the formation of large protein complexes that penetrate into the gel only slightly or not at all.
In summary, the presented electrode equipment is suited to utilize the advantages of miniaturized IEF systems, as described by Radola [ 6 ] ,using conventional horizontal IEF systems. The IEF procedure is fast and easy if flappable electrode holders without electrode strips are used. Graphite rods, which are cheap, readily available, and easy to process, can be used as electrodes as an alternative to platinized rods. An optimized method for easy sample application, as we describe here, is desirable if miniaturized IEF systems are used. Received July 3, 1990; in revised form February 5,1991
References Olsson, I., Axioe-Frederiksson, U.-B., Degermann, M. and Olsson, B., Electrophoresis 1988, 9, 16-22. Altland, K., in: Radola,B. J. and Graesslin, D. (Eds.), Elecrrofocusing and Isotachophoresis, Walter de Gruyter, Berlin 1977, pp. 295-301. PhastSystem Separation Technique File No. 100. Megow, D., Z . klin. Med. 1989, 44, 67-70. Megow, D. and Jacobasch, G., Electrophoresis 1990, II,65-69. Radola, B. J.,Kinzkofer, A. and Frey, M., in: Allen, R. C. and Arnaud, P. (Eds.),Electrophoresis '81,Walterde Gruyter,Berlin 1981,pp. 181189.
Electrophoresis 1991, 12, 378-380
D. Megow and 0.Jacobasch
A miniaturized electrode configuration for isoelectric focusing
Dirk Megow Gisela Jacobasch Institute of Biochemistry, Humboldt-University Berlin
An electrode configuration is described which allows fast isoelectric focusing (IEF) with conventional IEF systems. The equipment, which can be fixed on the cooling plate of a conventional IEF system, consists ofa base plate on which ffappable electrode holders are fastened. The handling is simple and needs only little time. Graphite rods are used as electrodes, thus avoiding the use of buffer strips Samples are applied with special applicator strips -permitting the analysis of up to 19 samples on a 50 X 40 mm polyacrylamide gel and up to 44 samples on a 100 X 70 mm gel. Only 30 min are needed for one IEF run.
The commercially available PhastSystem (Pharmacia), which permits short separation times, differs from conventional isoelectric focusing (IEF) units mainly by the simple handling of the IEF gels. It has a new electrode configuration, in which platinized titanium rods are placed directly on the gel surface, thereby avoiding the use of electrode strips. Samples are applied automaticallywith sample applicators [I]. We present an electrode configuration which makes it possible to carry out fast IEF on conventional horizontal IEF systems. Our electrode Configuration consists of
a base plate on which flappable electrode holders are fastened by sticking strips. The base plate can be fixed on the cooling plate of a conventional IEF system by adding a small amount of distilled water into the interspace. Two variants of the electrode configuration were used: IEF was carried out either with a two-electrode system (Fig. 1A) on 50 X 40 mm polyacrylamide gels or with a three-electrode system on 100 X 70 mm gels. The configuration with three electrodes consists of one central anode and two cathodes (Fig. 1B) as introduced by Altland [2]. We used graphite rods with a diameter of 3-5 mm as electrodes and placed them directly on the IEF gels, thus avoiding the use of buffer strips.
Y
/
Figure 2. Sample applicator: Small rubber squares are attached to a rigid support. Sample volumes of 0.5-1 pL are pipetted directly onto the squares, then for sample application the applicator is placed with the squares on the IEF gel.
2500 -
v
- 2.5
i
p
Figure I . Electrode equipment: Base plate with flappable electrode holders (fixed on the base plate by adhesive strips) and graphite electrodes: (A) Conventional configuration with electrodes and (B) three-electrode system with one central anode and two cathodes. (1) Flappable electrode holders with graphite electrodes, (2) polyacrylamide gel, (3) base plate.
~~
~~~
~
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
i
/ O 5
10
15
20
25
30
rnin
~~~~
Abbreviations: IEF, isoelectric focusing, PK, pyruvate kinase
mA,W
10.5
01 0
Correspondence: Dr. Dirk Megow, Institut fur Biochemie, Humboldt-Universitat, Wessische Str. 3-4, DO-1040 Berlin, Germany
I
Figure 3. Running conditions during IEF with a 50 X 40 mm polyacryl= 2500V, I,,, = 2 mA, amide gel, 3.5% Ampholine 3.5-10: V,,,
P,,
=2
w.
0173-0835/91/0505-0378 $3.50+.25/0
Elecfropkorcsrs 1991, 12, 378-380
A miniaturized electrode configuration for isoelectric focusing
379
PH 9-
7
-
7
3
I
0
I
I
10 20 30 gel length (mm)
Figure 4. pH gradient after IEF, determined with a pH marker mixture from Pharmacia. Running conditions as shown in Fig. 3. The corresponding IEF pattern is shown in Fig. 5A (lane 9).
Figure 5. IEF runs with 50 X 40 mm polyacrylamide gels. (A) 11samples and (B) 19 samples, respectively, were applied with the applicator strips shown in Fig. 2. (A) I500 ng (11, (2); 375 ng (31, (lo), (11); 94 ng (4); 31 ng (5); 7.5 ng (6); and 2 ng (7) of hemoglobin were applied. Detection limit was 31 ng with Coomassie Brilliant Blue staining. Pharmacia marker mixture (8) stored frozen at -18OC or (9) freshly dissolved, were applied at a loading of 100 ng perprotein. (B) Alternating application of hemoglobin (62 ng) and pH marker proteins (100 ng per each protein). Seventeen samples were applied, 2 lanes were left free. A mixing of the samples or a leaking into the blank lanes was not observed using the applicator strips.
IEFwas performed with an LKB2117Multiphorseparation unit and LKB 2197 power supply (Bromma, Sweden) on 200 pm thick polyacrylamide gels (5 O/oT, 4%C) with 3.5 O/o Ampholine 5-7 or 3.5-10 (LKB) and 12% glycerol on glass plates silanized with Silane A-174. Sample application was performed with special applicator strips where small rubber pieces are attached to a rigid support (Fig. 2). Sample volumes of 0.5-1 pL were pipetted directly onto the squares and then the applicator was placed with the side of the squares on the IEF gel. Under the chosen running conditions (Fig. 3) IEF was performed in about 30 min, comparable to run times obtained with the PhastSystem [3]. No excessive heat development of the graphite rods was observed. The pH gradient (Fig. 4), as estimated by a Pharmacia pH marker mixture, was nearly linear and not distorted by secondary electrode reactions of contaminations (of the graphite material) or reactions with the carrier ampholytes. However, some oxidative corrosion of the anodic graphite material was observed after repeated
use. This corrosion effect could be diminished by using especially pure graphite charges for electrodes. Nevertheless, an interchange of cathode and anode should be avoided because some electrode material can penetrate from the corroded electrode into the gel (not shown). Sample application was fast and easy using our applicator. Up to 19 samples could be analyzed on one 50 X 40 mm polyacrylamide gel (Fig. 5B). A mixing of the alternately applied hemoglobin and pH marker samples was not observed. No leaking of any protein into the blank lanes was detected. The detection limit for proteins was about 30 ng on staining with Coomassie Brilliant Blue (Fig. 5A). Other visualization procedures could also be applied and, as an example, Fig. 6 demonstrates the microheterogeneity of human erythrocyte pyruvate kinase (PK). PK was visualized by an indirect immunoassay with diaminobenzidine staining after immunofixation [4,51. IEF was carried out with the three electrode configuration where up to 44 samples can be applied on a 100 X 70 mm gel.
380
Electrophoresis 1991, 12, 378-380
D. Megow and G. Jacobasch
Figure 6. I E F of PK on a 100 X 70 mm polyacrylamide gel using the three-electrode system (Fig. 1B). PK was visualized by diaminobenzidine staining after immunofixation with a monospecific anti-PK serum and incubation with peroxidase-labeled second antibodies. Up to 44 samples can be analyzed with one gel. Differences in sharpness and intensity between the lanes are due to sample treatment with oxidative agents, partly resulting in the formation of large protein complexes that penetrate into the gel only slightly or not at all.
In summary, the presented electrode equipment is suited to utilize the advantages of miniaturized IEF systems, as described by Radola [ 6 ] ,using conventional horizontal IEF systems. The IEF procedure is fast and easy if flappable electrode holders without electrode strips are used. Graphite rods, which are cheap, readily available, and easy to process, can be used as electrodes as an alternative to platinized rods. An optimized method for easy sample application, as we describe here, is desirable if miniaturized IEF systems are used. Received July 3, 1990; in revised form February 5,1991
References Olsson, I., Axioe-Frederiksson, U.-B., Degermann, M. and Olsson, B., Electrophoresis 1988, 9, 16-22. Altland, K., in: Radola,B. J. and Graesslin, D. (Eds.), Elecrrofocusing and Isotachophoresis, Walter de Gruyter, Berlin 1977, pp. 295-301. PhastSystem Separation Technique File No. 100. Megow, D., Z . klin. Med. 1989, 44, 67-70. Megow, D. and Jacobasch, G., Electrophoresis 1990, II,65-69. Radola, B. J.,Kinzkofer, A. and Frey, M., in: Allen, R. C. and Arnaud, P. (Eds.),Electrophoresis '81,Walterde Gruyter,Berlin 1981,pp. 181189.
