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A multisample electrophoresis apparatus using vertical polyacrylamide gel slabs
ANALYTICAL
BIOCHEMISTRY
17,
A Multisample Vertical
32&326
(1966)
Electrophoresis
Apparatus
Polyacrylamide
Gel
HERBERT Max-Plan&-Znstitut
fib Received
Using
Slabs
TICHY Biologie, June
Tiibingen,
Germany
2, 1966
Electrophoresis in polyacrylamide gels, imroduced by Raymond and Wang (l), is widely used for the study of proteins in biological fluids. The advantage of this method is good separation in a short time. The analysis of a large series of samples by the disc electrophoresis method of Ornstein and Davis (2) with two gels and a discontinuous buffer system is, however, impeded because comparison between samples is difficult. Also, an apparatus allowing the simultaneous processing of many samples, like the one described by Matson (3), is not easy to handle. Since the apparatus of Raymond (4), using one buffer on a single vertical gel slab with 8 sample slots, has an insufficient capacit,y for the type of serial analysis to be carried ou.t in our laboratory, an apparatus for 49 samples was constructed that allows good separation and comparison among the samples. THE
APPARATUS
The apparatus is shown in Figure 1, with top and bottom separated to show the details. It is made entirely of Plexiglas, and consists of an upper (A) and lower (B) buffer reservoir each with a platinum wire (Pt) serving as electrode. The plate holder (C) is located between the buffer reservoirs; C holds between its side plates (E and G) the electrophoresis block (F), which consists of 7 gel plates and 8 cooling plates. Figure 1 shows only 2 cooling plates and 1 gel plate. The electrophoresis block (F) rests firmly on two rails (H) which are screwed onto (G). The pressure screw (D), whose pressure is evenly distributed by the side plate (E) onto the electrophoresis block (F), holds the cooling plates and gel plates firmly together. The plate holder (C) with the electrophoresis block (F) stands in the lower buffer reservoir (B) and is sealed with silicone grease into the recess of the upper buffer reservoir (A). The side plate (G) comes to rest between the wall of the recess in (A) and the projections (J), thus fixing the position of the entire holder (C) and the electrophoresis block (F). The opening in the upper 320
.G:LTISAJIPLE
GEL
321
ELECTROPHORESIE
-., I 5c I i-
FIG. 1. The electrophoresis apparatus (buffer reservoirs are separated from thr% plate holder and all dimensions are given in mm): (A) upper buffer reservoir. (B) lower buffer reservoir, (C) plate holder, (D) pressure screw, (E, G) side plates. (F) electrophoresis block with only 2 cooling plates ant1 1 gel plntr, (H) the rail, (I) projection, (Pt,) platinum electrode.
buffer reservoir (A) of 80 X 70 mm leaves uncovered only the upper side of the electrophoresis block (F). The cooling plates (Fig. 2A) are also made of Plexiglah. Each of the 8 cooling plates is sealed with two 0.5 mm plates of Plexiglas glued
322
HERBERT
TICHY
C
FIG. 2. The cooling plate (A), the gel mold (B) consisting of (a) glass plate with glass strips and (b) cover glass, and the slot former (C). 0 indicates marker tooth.
onto either side. The inlet and outlet of the coolant to each plate passes tube.
from a common
PREPARATION
OF THE
GEL
The gel mold is composed of 2 glass plates, 90 X 120 x 1 mm (Fig. 2B) used in photography as cover plates for slides. Before use all glass plates are dipped in a wetting medium (e.g., 0.5% Invitol, a photoGermany). This graphic wetting agent, A. Faber, Neu-Isenburg, prcqdure guarantees better removal of the gel from the glass plates after the run and allows proper cleaning of the plates from silicone grease .used for sealing. Each gIass pIate is laid horizontally on a block of plastic whose top surface is smaller than the glass plate. Two glass strips, 5 X 120 X 1 mm, are laid on the plates (Fig. 2B). There is no advantage in gluing the strips onto the plates. The material for the glass strips is cut off a plate. Gel solutin (10 ml per plate), made according to Raymond and Wang (l), is poured onto the plates with a pipet.
Surface tension prevents leaking of the gel solution. Another glass plate is then put on top of the gel-bearing plat.e, care being taken not to trap any air bubbles. The slot former (Fig. 2C), made of Plexiglas 1 mm t.hick, is then inserted from one side between the glass plates into the gel solution and polymerization is allowed to proceed. The opposite side remains uncovered and the gel remains unpolymeri& to :I depth of 2-3 mm. This has no adverse effects. PROCEDURE
The cooling plates are inserted into the plate holder and inlet and outlet tubes are connected with the common tubes for the coolant. Before each run the cooling plates are precooled for at least, 15 min. The plate holder is placed into the lower buffer reservoir to which enough buffer is then added to cover the rails (Fig. 1H) and the electrode. Then the gel plates are inserted each between two cooling plates, after removing the slot former and leaving the gel and glass plates together. The slot made by the marker toot,h 0 (Fig. 2C) must always point to the same side. After placing all gel plates int,o the holder, th@ electrophoresis block is tightly pressed together wit,h the screw (Fig. 1D). A rim of medium-viscosity silicone grease is applied round the upper edge of the electrophoresis block. The silicone grease serves to seal the electrophoresis block to the upper reservoir. Then the upper’ buffer reservoir is pressed onto the plate holder. With a drawn-out, OI fine-tipped pipet the sample slots are filled with buffer, and then t,hr> samples are applied with Wcrocaps (Drummond Scientific Co.j. If the samples are colorless they can be st’ained with bromphenol blue. If they are not dense enough to sink to the bottom of the slot their densi@ can be increased by adding sucrose. The application of t.he 49 samples takes 30-60 min. After all samples are applied, the upper buffer reservoir is gently filled with buffer so that it stands 10 mm over the plates. For the dimensions given, a tot,al of 3 liters of buffer is necdec\. Then the cllrrent is swit,ched on. RESULTS
The best buffer for the separation of the hemolymph proteins of larvae is the 0.1 JJ tris-EDTA-boric acid buffer of Aronsson and Griinwall (5) with a pH of 8.6-8.8. A 107% acrylamide gel was produced from Cyanogum 41 (1) : 10 gm of Cyanogum 41 was dissolved in 100 ml of t,he buffer; to catalyze gel formation 0.6 ml of dimethylaminopropionitrile (DMAPN) and 0.6 ml of a freshly made 10% solution of ammonium persulfate WIY added. Chironomus tentans (Diptera)
324
HERBERT
FIG.
3. Separation
four gel plates of
Other conditions
tivo
of hemolymph different runs.
from For
TICHT
Chironomus tentans (Dipteral--twice experimental conditions see text.
were: 400 V and a current of 150 mA; normal
room
kmperat,u-e; tap-water coolant at 8”, kept constant for a run of 2 hr at 300 ml/min. At t,he end of a run the gel plates are removed from the apparatus
.\fUL’lXhMPLE
GEL
ELEC’TROPHORESIS
325
and one glass plate is lifted off; the gel resting on the remaining glass plate is stained with amido black 1OB and destained electrophoreticall;v in 2% acetic acid. J!‘our plates 0i Figure 3 shows the results obtained by thlL‘I: m&od. two different runs are shown. Each sample is derived from a larva in a different state of development and from various locations. From each larva, 2 pll hemolymph was used containing 15-70 pg protein, depending on the larval stage. The wet gel can be analyzed with any t,ype of densitometer. To preserve the gel slabs they are allowed to shrink in 50% ethanol to half their size. Then they are covered with cellophane, put between filter papers, and dried under low pressure in a drying oven at 100”. The dried gels can be swollen back to t.heir original Pize by immersion in mater. DISCUSSIOK The electrophoresis apparatus described allows the &llultaneous analysis of 49 samples. This is accomplished on 7 polyacrylamide gel slabs of 1 mm thickness. The capacity can easily be doubled b3 increasing the number of samples per plate. Since the gel slabs are cooled it is possible to use up to 600 V without loss of resolution. In horizontal gel electrophoresis where the samples are applied by the filter-paper method (1, 6) one can often see tailing from the start. This tailing has not been observed with the present met,hod. Furthermore the sample volume is known exactly and losses by retention on paper cannot occur. The load for opt.imal resolution was 20-50 pg Chi~o~~~.s hemolymph protein. Good results were obtained with sample volumes between 0.2 and 20 ~1. The sample should be in a relatively concentrated solution to allow the application of sufficient protein in a volume within the optimal range. For t,he present purpose the separation was as good a> the one obtained by disc electrophoresis. The main advantage of this apparatus is the possibility of comparing different samples with each other exactly, samples of one plate with each other and also with samples on different plates. High reproducibility is also achieved bct.ween different. runs if care is taken to maintain uniform conditions. 8u~h :t romparison in exemplifirrl hy Figure 3. SUMMARY -111 electrophoresis apparatus for 49 samplea, using vertical polyscrylamide gel slabs, is described. Good separation and high reproducibility arr achieved which RIIOW ior :t dire& caomparison among ;I large tlllrnl~et* c~f rnmplrr.
326
HERBERT
TICHY
ACKNOWLEDGMENTS I wish to thank Professor !V. Beermann for his interest for discussion and help in translating this manuscript.
and Dr. I. B. Dawid
REFERENCES 1. RAYMOND, S., AND WANG, Y., Anal. Biochem. 1, 391-396 (1960). 2. ORNSTEIN, L., AND DAVIS, B. J., “Disc Electrophoresis,” preprint, Distillation Products Industries, Rochester, N. Y., 1962. 3. MATSON, C. F., Anal. Biochem. 13, 294-304 (1965). 4. RAYMOND, S., Clin. Chem. 8, 455-470 (1962). 5. ARONSSON, T., AND GR~NWALL, A., &and. J. Clin. Lab. Invest. 9, 338 (1957). 6. SMITHIES, O., Biochem. J. 61, 629-641 (1955).
BIOCHEMISTRY
17,
A Multisample Vertical
32&326
(1966)
Electrophoresis
Apparatus
Polyacrylamide
Gel
HERBERT Max-Plan&-Znstitut
fib Received
Using
Slabs
TICHY Biologie, June
Tiibingen,
Germany
2, 1966
Electrophoresis in polyacrylamide gels, imroduced by Raymond and Wang (l), is widely used for the study of proteins in biological fluids. The advantage of this method is good separation in a short time. The analysis of a large series of samples by the disc electrophoresis method of Ornstein and Davis (2) with two gels and a discontinuous buffer system is, however, impeded because comparison between samples is difficult. Also, an apparatus allowing the simultaneous processing of many samples, like the one described by Matson (3), is not easy to handle. Since the apparatus of Raymond (4), using one buffer on a single vertical gel slab with 8 sample slots, has an insufficient capacit,y for the type of serial analysis to be carried ou.t in our laboratory, an apparatus for 49 samples was constructed that allows good separation and comparison among the samples. THE
APPARATUS
The apparatus is shown in Figure 1, with top and bottom separated to show the details. It is made entirely of Plexiglas, and consists of an upper (A) and lower (B) buffer reservoir each with a platinum wire (Pt) serving as electrode. The plate holder (C) is located between the buffer reservoirs; C holds between its side plates (E and G) the electrophoresis block (F), which consists of 7 gel plates and 8 cooling plates. Figure 1 shows only 2 cooling plates and 1 gel plate. The electrophoresis block (F) rests firmly on two rails (H) which are screwed onto (G). The pressure screw (D), whose pressure is evenly distributed by the side plate (E) onto the electrophoresis block (F), holds the cooling plates and gel plates firmly together. The plate holder (C) with the electrophoresis block (F) stands in the lower buffer reservoir (B) and is sealed with silicone grease into the recess of the upper buffer reservoir (A). The side plate (G) comes to rest between the wall of the recess in (A) and the projections (J), thus fixing the position of the entire holder (C) and the electrophoresis block (F). The opening in the upper 320
.G:LTISAJIPLE
GEL
321
ELECTROPHORESIE
-., I 5c I i-
FIG. 1. The electrophoresis apparatus (buffer reservoirs are separated from thr% plate holder and all dimensions are given in mm): (A) upper buffer reservoir. (B) lower buffer reservoir, (C) plate holder, (D) pressure screw, (E, G) side plates. (F) electrophoresis block with only 2 cooling plates ant1 1 gel plntr, (H) the rail, (I) projection, (Pt,) platinum electrode.
buffer reservoir (A) of 80 X 70 mm leaves uncovered only the upper side of the electrophoresis block (F). The cooling plates (Fig. 2A) are also made of Plexiglah. Each of the 8 cooling plates is sealed with two 0.5 mm plates of Plexiglas glued
322
HERBERT
TICHY
C
FIG. 2. The cooling plate (A), the gel mold (B) consisting of (a) glass plate with glass strips and (b) cover glass, and the slot former (C). 0 indicates marker tooth.
onto either side. The inlet and outlet of the coolant to each plate passes tube.
from a common
PREPARATION
OF THE
GEL
The gel mold is composed of 2 glass plates, 90 X 120 x 1 mm (Fig. 2B) used in photography as cover plates for slides. Before use all glass plates are dipped in a wetting medium (e.g., 0.5% Invitol, a photoGermany). This graphic wetting agent, A. Faber, Neu-Isenburg, prcqdure guarantees better removal of the gel from the glass plates after the run and allows proper cleaning of the plates from silicone grease .used for sealing. Each gIass pIate is laid horizontally on a block of plastic whose top surface is smaller than the glass plate. Two glass strips, 5 X 120 X 1 mm, are laid on the plates (Fig. 2B). There is no advantage in gluing the strips onto the plates. The material for the glass strips is cut off a plate. Gel solutin (10 ml per plate), made according to Raymond and Wang (l), is poured onto the plates with a pipet.
Surface tension prevents leaking of the gel solution. Another glass plate is then put on top of the gel-bearing plat.e, care being taken not to trap any air bubbles. The slot former (Fig. 2C), made of Plexiglas 1 mm t.hick, is then inserted from one side between the glass plates into the gel solution and polymerization is allowed to proceed. The opposite side remains uncovered and the gel remains unpolymeri& to :I depth of 2-3 mm. This has no adverse effects. PROCEDURE
The cooling plates are inserted into the plate holder and inlet and outlet tubes are connected with the common tubes for the coolant. Before each run the cooling plates are precooled for at least, 15 min. The plate holder is placed into the lower buffer reservoir to which enough buffer is then added to cover the rails (Fig. 1H) and the electrode. Then the gel plates are inserted each between two cooling plates, after removing the slot former and leaving the gel and glass plates together. The slot made by the marker toot,h 0 (Fig. 2C) must always point to the same side. After placing all gel plates int,o the holder, th@ electrophoresis block is tightly pressed together wit,h the screw (Fig. 1D). A rim of medium-viscosity silicone grease is applied round the upper edge of the electrophoresis block. The silicone grease serves to seal the electrophoresis block to the upper reservoir. Then the upper’ buffer reservoir is pressed onto the plate holder. With a drawn-out, OI fine-tipped pipet the sample slots are filled with buffer, and then t,hr> samples are applied with Wcrocaps (Drummond Scientific Co.j. If the samples are colorless they can be st’ained with bromphenol blue. If they are not dense enough to sink to the bottom of the slot their densi@ can be increased by adding sucrose. The application of t.he 49 samples takes 30-60 min. After all samples are applied, the upper buffer reservoir is gently filled with buffer so that it stands 10 mm over the plates. For the dimensions given, a tot,al of 3 liters of buffer is necdec\. Then the cllrrent is swit,ched on. RESULTS
The best buffer for the separation of the hemolymph proteins of larvae is the 0.1 JJ tris-EDTA-boric acid buffer of Aronsson and Griinwall (5) with a pH of 8.6-8.8. A 107% acrylamide gel was produced from Cyanogum 41 (1) : 10 gm of Cyanogum 41 was dissolved in 100 ml of t,he buffer; to catalyze gel formation 0.6 ml of dimethylaminopropionitrile (DMAPN) and 0.6 ml of a freshly made 10% solution of ammonium persulfate WIY added. Chironomus tentans (Diptera)
324
HERBERT
FIG.
3. Separation
four gel plates of
Other conditions
tivo
of hemolymph different runs.
from For
TICHT
Chironomus tentans (Dipteral--twice experimental conditions see text.
were: 400 V and a current of 150 mA; normal
room
kmperat,u-e; tap-water coolant at 8”, kept constant for a run of 2 hr at 300 ml/min. At t,he end of a run the gel plates are removed from the apparatus
.\fUL’lXhMPLE
GEL
ELEC’TROPHORESIS
325
and one glass plate is lifted off; the gel resting on the remaining glass plate is stained with amido black 1OB and destained electrophoreticall;v in 2% acetic acid. J!‘our plates 0i Figure 3 shows the results obtained by thlL‘I: m&od. two different runs are shown. Each sample is derived from a larva in a different state of development and from various locations. From each larva, 2 pll hemolymph was used containing 15-70 pg protein, depending on the larval stage. The wet gel can be analyzed with any t,ype of densitometer. To preserve the gel slabs they are allowed to shrink in 50% ethanol to half their size. Then they are covered with cellophane, put between filter papers, and dried under low pressure in a drying oven at 100”. The dried gels can be swollen back to t.heir original Pize by immersion in mater. DISCUSSIOK The electrophoresis apparatus described allows the &llultaneous analysis of 49 samples. This is accomplished on 7 polyacrylamide gel slabs of 1 mm thickness. The capacity can easily be doubled b3 increasing the number of samples per plate. Since the gel slabs are cooled it is possible to use up to 600 V without loss of resolution. In horizontal gel electrophoresis where the samples are applied by the filter-paper method (1, 6) one can often see tailing from the start. This tailing has not been observed with the present met,hod. Furthermore the sample volume is known exactly and losses by retention on paper cannot occur. The load for opt.imal resolution was 20-50 pg Chi~o~~~.s hemolymph protein. Good results were obtained with sample volumes between 0.2 and 20 ~1. The sample should be in a relatively concentrated solution to allow the application of sufficient protein in a volume within the optimal range. For t,he present purpose the separation was as good a> the one obtained by disc electrophoresis. The main advantage of this apparatus is the possibility of comparing different samples with each other exactly, samples of one plate with each other and also with samples on different plates. High reproducibility is also achieved bct.ween different. runs if care is taken to maintain uniform conditions. 8u~h :t romparison in exemplifirrl hy Figure 3. SUMMARY -111 electrophoresis apparatus for 49 samplea, using vertical polyscrylamide gel slabs, is described. Good separation and high reproducibility arr achieved which RIIOW ior :t dire& caomparison among ;I large tlllrnl~et* c~f rnmplrr.
326
HERBERT
TICHY
ACKNOWLEDGMENTS I wish to thank Professor !V. Beermann for his interest for discussion and help in translating this manuscript.
and Dr. I. B. Dawid
REFERENCES 1. RAYMOND, S., AND WANG, Y., Anal. Biochem. 1, 391-396 (1960). 2. ORNSTEIN, L., AND DAVIS, B. J., “Disc Electrophoresis,” preprint, Distillation Products Industries, Rochester, N. Y., 1962. 3. MATSON, C. F., Anal. Biochem. 13, 294-304 (1965). 4. RAYMOND, S., Clin. Chem. 8, 455-470 (1962). 5. ARONSSON, T., AND GR~NWALL, A., &and. J. Clin. Lab. Invest. 9, 338 (1957). 6. SMITHIES, O., Biochem. J. 61, 629-641 (1955).