ANALYTICAL BIOCHEMISTRY 81, 10% 117 (1977)

An Apparatus for the Simultaneous Performance of Polyacrylamide-Gel Electrophoresis at

Multiple pHs

CATHERINE Bur,’ VINCENT GALEA,~ AND ANDREAS CHRAMBACH

Received November lg. 1976: accepted February 8. 1977

Optimization of the operative pH of polyacrylamide-gel electrophoresis can be carried out on a single workday by a single operator using a multichamber PAGE apparatus. The apparatus was shown to be capable of simultaneous gel electro- phoresis at up to 10 different pH values.

Resolution between proteins in polyacrylamide-gel electrophoresis (PAGE) (1) depends on differences between the molecular net charges of the species to be separated, in addition to the exploitation of differences in molecular geometry. The efficient use of molecular net charge dif- ferences for fractionation again depends on operation at a pH at which the net charge difference between the two species is maximal. Usually, this difference increases as the absolute values of molecular net charges are decreased, i.e., as one approaches the isoelectric point of the two species. The limit to which such approximation toward the isoelectric point can be applied in PAGE is set by the need to maintain a finite mobility. When PAGE is conducted in continuous buffers, the only lower limit of mobility is set by the practical requirement to terminate the electro- phoresis experiment within a reasonable time period. In applying PAGE in multiphasic buffer systems (multiphasic zone electrophoresis, MZE) (3, another limit to the lowering (or increase) of pH toward the isoelectric point is set by the need to stack the protein, i.e., to achieve a constituent mobility for the protein equal to or more than that of the trailing constituent of the multiphasic buffer system (designated as the lower stacking limit) (1).

Thus, when one is faced with the need to optimize the pH of MZE for a particular protein fractionation problem, the first step is a step-by-step

’ Guest worker. o Hoefer Scientific Instruments. San Francisco. California 94107.

108

TEN-pH PAGE APPARATUS 109

variation in the pH of stacking gels, the purpose of which is to ascertain the minimal pH, at a very low value of the lower stacking limit, at which the protein of interest migrates within the stack [p. 64 of Ref. (1): Fig. 1A of Ref. (3)]. Once this pH is delineated, a second similar experiment is carried out in which the lower stacking limit is increased stepwise to ascertain the maximal value of the lower stacking limit compatible with the stacking of the protein of interest [Fig. IB of Ref. (3)].

Both experiments, the determination of the optimal stacking pH and the determination of the optimal lower stacking limit, involve PAGE in multiple buffers, usually 5- 10 different buffers (although in the second pH optimization experiment the buffer constituents of each are the same, and gels could be subjected to PAGE in the same apparatus, if one could disregard differences in conductance and Joule heating between gels operative in the various “subsystems”). To date, therefore, this type of experiment had to be carried out in 5-10 different PAGE ap- paratus units simultaneously, hardly a possibility in most laboratories not equipped with such a wealth of apparatus, or it had to be conducted successively, requiring an excessive amount of time. Either of the two approaches seemed unduly laborious.

We have, therefore, designed a PAGE apparatus for the separate, simultaneous performance of PAGE in single stacking gels operating at up to 10 different pH values. This report details the design. construction, and performance of the apparatus.

CONSTRUCTION

1. Electrophoresis trrlit (Fig. I) The unit is composed of five parts which shall be described in order of alignment from top to bottom. It is made of polycarbonate (parts A, C. E) of 1.5mm wall thickness and Plexiglas (parts B, D) of IO-mm wall thickness.

Part A. the top compartment of the unit, serves as an upper buffer reservoir. Al is a tube of 4%mm i.d. A2 is a lock plate perforated at the corners. The lock plate is positioned at a distance of 4-7 mm from the bottom of tube Al. All lock plates (in this unit and the others) are equally aligned with respect to both the horizontal and the vertical axes of the unit. For ease of alignment, lock plates have been designed rec- tangularly. The shorter sides of the rectangular plate should face the front and back of the unit. Screw connections between lock plates should be made fimly and with maintenance of alignment, using screws of 1 5/8-in. length inserted through holes A3 to provide a tight connection between parts A and B. Screws are inserted facing downward. To ensure the alignment, lock plates should be permanently glued to their respective tubes by epoxy adhesive.

Part B connects by press-fit (O-ring) to the bottom of compartment A. Bl is a male banana plug in contact with a platinum wire electrode ex-

110 BUI. GALEA. AND CHRAMBACH

A)

a b k--51---7

A2

B)

side view bottom view

-644 side view top view

FIG. 1. Electrophoresis unit of the apparatus. (a) Component parts: (b) assembled unit. (A) upper buffer reservoir. (C) coolant chamber. (E) lower buffer reservoir. A gel tube (not shown) is placed between A and C through rubber grommets B4 and D4. Platinum wire electrodes extend into parts A and C through banana plugs Bl and Dl. Dimensions are in millimeters.

tending to the bottom of buffer chamber B (the interior of tube B above grommet B4). A corresponding female banana plug F4 is located on mount- ing board Fl of the apparatus (Fig. 2). Rubber O-rings B2 (No. 2-31) serve to connect part B with parts A and C. B3 is the coolant outlet

TEN-pH PAGE APPARATUS 111

FIG. 2. Mounting board for 10 electrophoresis units, containing a power inlet (F2, F3), a common power line connected to female banana plugs F4 and FS. and central coolant inlet and outlet tubes (F6. F7) which are connected to each of the units via tubes FS and F9. Dimensions are in millimeters.

port (&mm i.d., 9-mm o.d.1 connected to female quick-disconnect F13 by vinyl tubing F8 (Fig. 3). Outlet port B3, if screwed into part B too tightly, prevents a good O-ring fit between parts A or C and B. It must, therefore, be loosened prior to connecting these parts and should be retightened after connection has been made. B4 is a rubber grommet (S-mm i.d.1 at the center of part B. The gel tube is held in place between grommets B4 and D4. BS is a semicylindrical hole in the wall of B extending from the bottom of B to outlet port B3. On assembly, care must be taken to orient BS downward.

Part Cl is the coolant reservoir. During electrophoresis, most of the length of the gel tube is immersed in Cl. B is inserted into the top of C. C2 is a lock plate fastened to lock plate A2 by screws. The lock plate should be attached within 4-7 mm of the top of tube Cl. C3 is the lock plate attached by screws to E3. The screws face upward and pass through holes C4 in the lock plate.

112 BUI, GALEA, AND CHRAMBACH

FIG. 3. Coolant supply lines to the units. Central coolant inlet and outlet tubes FlO and Fl? feed into each of the units via tubing connections F8 and F9.

Part D fits into the bottom of compartment C. Part DI is a male banana plug connected to a platinum wire extending to the bottom of buffer chamber D (the interior of tube D below grommet D4). This plug fits into a female banana plug inserted into mounting board Fl of the apparatus (Fig. 2). D2 are O-rings (No. 2-31) connecting D to C and E. D3 is the coolant inlet port (4.5mm i.d., 7-mm 0.d.). It connects by vinyl tubing to female quick-disconnect D6 (Fig. 4). D4 is a grommet into which the gel tube is inserted. D5 is a semicylindrical hole in the wall of D which must face upward to avoid leakage.

Part E is the bottom part of the unit. D is connected by O-ring D2 with part E. El forms the lower buffer reservoir. It is similar to part A except that it is sealed at the bottom. Lock plate E2 is fastened to C3 by screws, facing upward, which pass through the four holes E3 and C4.

2. Assembled apparatus. Part Fl is the mounting board of the apparatus (Fig. 2). Male banana plugs F2 and F3 connect to the power supply. The individual units are attached to the mounting board by connection between the female banana plugs (F4 and F5) and the male banana plugs on the units (BI and Dl, Fig. 1). Coolant enters through port F6 into the PVC coolant inlet tube FIO, and exits via port F7 from PVC coolant outlet tube F12 (Fig. 3). During operation, cover plate G (Fig. 5) is placed on top ofthe apparatus for safety and to prevent contamination.

Each unit is cooled by connection of PVC central coolant inlet tube F10 (%-in. o.d., S-in. i.d.) to coolant inlet port D3 via %-in. i.d., %6-in. o.d. vinyl tubing (Fig. 4). Coolant outlet port B3 on each unit is connected to central PVC coolant outlet tube F12. These tubing connections D3- F10 and B3-F12 contain quick-disconnects (A. H. Thomas No. 9583-M14)

TEN-pH PAGE APPARATUS 113

f=Y PVC /

F13 83 lfemalel

F13 Cmalel

FS --f /

-y=j

D6 D3 Ifemalel

FIG. 4. Schematic coolant flow diagram. Coolant enters from a thermostated bath through central coolant inlet tube F6 into the unit shown on the right. Coolant exits from the unit into central coolant outflow tube F12. Vinyl tubing connections are designated as F8 and F9. quick-disconnects as Fl3 and B6 and Dh.

D6-F13 and B6-F13, respectively. Central coolant outlet tube F12 is in- stalled in the mounting board at an elevation of 2 cm above central coolant inlet tube FlO. Connecting tubes F8 and F9 are attached to the central coolant tubes by male polyethylene tubing connectors Fll, which are permanently threaded into tubes FlO and F12. The assembled ap- paratus, with gel tubes in place. is shown in Fig. 5.

OPERATION

Polynzcrixztiorl of’strrcking gt~ls. Stacking gels (5%T. 15%C,,,,) in 10 MZE systems (phases BETA) of interest were polymerized in a single analytical PAGE apparatus [Fig. 13 of Ref. (l)]. The apparatus and procedure for PAGE were as described previously ( 1). The compositions of the representative MZE buffer systems as used in this study are given in Appendix I. Gel volumes were 1.7-2.0 ml per tube (Pyrex. 7-mm o.d.. 5-mm i.d.) of 6-in. length.

The lower buffer reservoir E 1 of each unit was filled with the appropriate lower buffer of each MZE buffer system. Gel tubes (with the Parafilm seal removed) of each designated MZE buffer system were inserted through grommets B4 and D4 into the particular unit assigned to this system. For tube insertion, part B was removed from C: the tube was then passed through grommet B4. Then the bottom of the tube, attached to B. was inserted into grommet D4, and the tube was pushed downward into lower buffer reservoir El until just a few millimeters of tube length remained in the upper buffer reservoir. Screw connection between

114 BUI, GALEA. AND CHRAMBACH

TEN-pH PAGE APPARATUS 115

lock plates E2 and C3 remained tightened while those between A2 and C2 had to be tightened after insertion of the tube. When the unit was connected, lower buffer in excess of the volume needed to fill El flowed out through hole D6. If at any time during electrophoresis the lower buffer level should drop, lower buffer can be introduced into the reservoir El through hole D6 by syringe. Units were connected to mounting board Fl via banana plugs Bl and Dl. Quick-disconnects B6 and D6 were severed. Coolant flow was stopped, and quick-disconnects F13 were separated. Tubes F8 and F9 were then connected by quick- disconnects B6 and D6 to coolant outlet and inlet ports B3 and D3 (Fig. 4). Coolant flow was reinitiated at a flow rate of not less than 1.0 literimin. Air bubbles were cleared from all the lines. The upper buffer reservoirs Al were filled with the appropriate upper buffers. Air bubbles were removed from the tops of the gels by insertion of a Pasteur pipet into each tube. Samples were applied to the gel surface by Pasteur pipet and pipet control (Curtin Matheson Scientific No. 059-709). using the procedure previously described (1). Coverplate G was placed onto mounting board F. Banana plugs F2 and F3 were connected to the power supply (Kepco BHK 2000-O.lM, Flushing, New York).

Electrophoresis was then conducted at 1 mA/tube and discontinued when the stack, marked by a tracking dye, had reached two-thirds of the gel length in one of the tubes. The single unit containing this tube was withdrawn, after momentary discontinuation of coolant flow, severance, and reconnection of the appropriate quick-disconnects. Electrophoresis was continued for the remaining units as soon as possible. Coolant in the separated unit was drained. Upper buffer was poured out, with holes D6 closed by finger to prevent loss of lower buffer. Screws connecting A2 and C2 were removed, parts A and B were separated from C and the gel tube was pulled out of the grommet. The gel was removed from the tube, fixed and stained as described for stacking gels previously (1,3).

PERFORMANCE

1. Stacking of BSA at the 10 pH values was carried out in a single experiment, by a single operator, in a period of 7-8 hr inclusive of the time needed for cleaning and reassembly of the apparatus. The results of such a simultaneous stacking analysis at 10 pH values are given in Fig. 6. BSA stacks at pH values ranging from 6.12 to 10.45, as evidenced by the sharp protein zone boundaries which did not widen with increasing time of electrophoresis and by the identity in migration distances relative to gel length between the tracking dye, bromophenol blue, on the unstained gels and the stained protein band on the same gels after fixation and staining.3

’ The typical appearance of a stacked protein zone as compared with a zone of unstacked protein is shown in Fig. 11 of Ref. (1) and Fig. 1 of Ref. (3).

116 BUI, GALEA. AND CHRAMBACH

0 ?e-+

1830 1906 1933 1964.4 1935 2254 2333 2365 2860 2950 MZE buffer system

6.12 6.43 7.10 7.24 7.42 7.81 7.97 8.10 9.63 10.45 pti IZETAI

FIG. 6. Stacking gels (5%T, 1F’C ,c I,ATU) depicting BSA stacked simultaneously at 10 pH values. Gels after fixation and staining are shown. Compositions of the MZE buffer systems corresponding to the 10 pH values are specified in Appendix I.

2. During electrophoresis of 10 stacking gels, the temperature inside coolant chamber Cl was measured within a gel tube containing water above a gel plug, using an electronic multichannel telethermometer (TRI-R Model TGL, Serial No. H541). The approximate time needed to cool the apparatus to a steady-state temperature of 0..5-3.5”C above bath temperature within each unit was 1 hr of electrophoresis. At the coolant flow rates used, the steady-state temperature on the side of the coolant inflow (front face of the mounting board) was within 0.X of the bath temperature. On the distal side of the mounting board, the temperature varied by 0.5 to 3.5”C from the bath temperature. Such temperature variations appear negligible for the electrophoresis of stacking gels for which the apparatus was designed.

CONCLUSIONS

An apparatus allowing for pH optimization in PAGE by a single operator during a single workday has been constructed, tested, and found to be capable of simultaneous PAGE analysis at up to 10 different operative pHs. Temperature control in the apparatus is sufficient to allow for such application at an operative temperature of 0-4°C.

ACKNOWLEDGMENTS

We thank Ms. Estelle Watts and Ms. Donna Jackson. Medical Arts and Photography Branch, Division of Research Services, NIH. for their expert graphic work in preparing the figures and Mr. Wes Pearson, of the same branch, for the photography.

TEN-pH PAGE APPARATUS 117

REFERENCES

I. Chrambach, A.. Jovin, T. M., Svendsen. P. J.. and Rodbard. D. (1976) ill Methods of Protein Separation (Catsimpoolas. N.. ed.), pp. 27-134. Plenum Press. New York.

2. Jovin, T. M. (1973) Bioc.l~c~r~zi.\r,. 12. a) 871-878: 879-889: 890-898. 3. Chrambach. A.. and Skyler. J. D. irl XXII Annual Colloquium on Protidex of the

Biological Fluids (Peeters. H.. ed.). pp. 701-713. Pergamon Press. Oxford.