INSTRUMENTATION
Advisory Panel Jonathah W. Amy Jack W. Frazer G. Phillip Hicks
Donald R. Johnson Charles E. Klopfenstein Marvin Margoshes
H a r r y L. Pardue Ralph E. Thiers William F. Ulrich
Electrofocusing in Gels DANIEL WELLNER Department of Biochemistry, Cornell University Medical College New York, N. Y. 10021
Gel electrofocusing provides the enzymologist, the protein chemist, the geneticist, the immunologist, and the clinician with a versatile supplementary research tool for the analysis and characterization of proteins and peptides. Its simplicity, its high resolving power, and its ease of combination with other techniques indicate that the number of applications to which it will be put will greatly increase in the near future HE TECHNIQUE of gel elect,rofociisT i n g is one of tlie most promising new methods for the analysis and characterization of enzymes, hormones, and ot’her ampholyt,es of biological interest.. I t is equal or superior in sensitivity, resolution, and simplicity to other analyt’ical methods commonly employed for the study of proteins. Furthermore, because it separates compounds on t,he basis of differences in isoelectric point, it provides additional information not readily obtainable by other methods. Therefore, while electrofocusing is not likely to displace such methods as electrophoresis. chromatography, or iiltrncentrifupation. it represents a valiiable Pupplementary resenrch tool. I n thiP article, the principle of the method i d 1 be outlined briefly, the csperimental technique will be dcscribed, and some esnmples of its i i s r will be given.
Principle of Method
In an electric field, proteins and other ampholytes move toward t,he cathode in solutions more acidic than their isoelectric point and toward the anode in solutions more basic than their isoelectric point’. At t,heir isoelectric point (vdien pH = p I ) they carry no net charge and therefore do not migrate. When a protein is added to a solution containing a p H gradient, and when an electric potential is applied to such a solution, with the m o d e on the acidic side and the cathode on the basic side, the protein molecules migrate toward tlmt region of the gradient where the pH equals their isoelectric point. If the p H gradient were stable and if convection and diffusion were absent. the protein would become concentrated in a sharp stationary zone, its position depending 011 the isoclectric point of the protein. -411 early attempt to esploit, this principle for the separation and concentration of proteins was made by Kolin ( 1 ) in 1954. H e used a gradient formed between two buffers of different p H and. at the same time, a sucrose density gradient to prevent convective remising of the separnted proteins. Although good wparations were ncliieved in that systen. the sharpness of tlie focusing was limited by the migration of the buffer ions in the electric field and the conseqiient inst.3bility of the pH gradient. This problem was overcome by Svensson ( 2 ) and Vesterberg and Svensson ( 3 ) b y the use of a “natural” pH gradient-i.e., n gradient formed iiiider the infliience of an electric current a n d stable in the presence of the current. This was accomplished by wing “carrier ampholyt~es”consisting of a mixtiire of many compounds of low molecular weight (- from their isoelectric position bring exactly coiinteracted by electrophoretic migration in tlie opposite clirect ion.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
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Instrumentation
for focusing t o be complete on the small scale used in gels than in the column procedure. Greater resolution may also be achieved in gels because it is possible to avoid the mixing of closely spaced bands which takes place when gradient solutions are transferred to a fraction collector. I n addition, the convective remixing which occurs in sucrose gradients when focusing produces excessive local concent>rations of proteins does not take place in gels. Finall\-, gel electrofocusing makes it simple to analyze many samples simultaneously and under identical conditions for comparative purposes.
Experimental Procedure
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Figure 1. focusing
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Apparatus for gel electro-
When proteins are introduced in such a system, either before or after the gradient is established, t'hey are concentrated or focused in sharp zones. Their isoelectric points may be determined wit,h accuracy simply by measuring the pH of these zones. In t,his way, proteins differing in isoelect,ric point, by as little a.s 0.02 pH unit, can be resolved from one another (5'). I n 1968, not long after suitable carrier ampholytes became commercially available (several ranges of p I are available from LKB Produkter -4B, Bromma, Sweden), procedures for performing electrofocusing in polyacrylamide gels were developed independently in many laboratories (5-14). Polyacrylamide gel has several important advantages as an anticonvection medium over sucrose denshy gradients, One of these is that it allows separat'ions to be carried out on a much smnller scale, thereby making it. possible t o analyze smaller samples and to work with smaller amounts of the relatively expensive ampholytes. Another advantage is that it, makes unnecessary the use of elaborate jacketed glass columns. Instead, experiments can be carried out in a very simple apparatus of the type used for gel el~ctrophoresis,as described below. Furthermore, less time is required
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ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
A simple apparatus which has been used successfully for gel electrofocusing in this laboratory is shown diagrammatically in Figure 1. It is similar to t h a t described by Davis ( 1 5 ) for disc electrophoresis. The apparatus consists of two cylindrical Plexiglas containers whose covers are fitted with platinum wire electrodes. The bottom of the upper container has a number of circular openings symmetrically arranged along its circumference. These Lire fitted with rubber stoppers through nhich a 6-mm i.d. glass tube has been inserted. When an experiment involves only one or a few samples, the unused openings may be closed with solid stoppers. The bottom container has a corresponding number of openings in its cover. Glass tube.s of various lengtlis may be used, the height of the upper container being yaried accordingly by raising or lowring its support on a ring stand. Since electrofocusing concentrat'es proteins into sharp zones, the sample may he uniformly distributed throughout the gel a t the start of the experiment. Thus, it is possible t o polymerize the acrylamide in a solution containing both the carrier ampholytes and the sample to be analyzed. When this procedure is adopted, photopolymerization with riboflavin as catalyst is the preferred method of forming the gel. This avoids the possibility of alteration of protein samples which may occiir in the presence of persulfate. Photopolymerization is a mild procedure, 30 min a t room temperature under ordinary laboratory overhead lights usually being sufficient for the ge1 to form. Gel formation is sometimPs facilitated by removing dissolved oxygen from the solution under reduced p r e-ure. x I n some cases, however, i t is not possible to prepare satisfactory gels b y phot opolymerization, For example,
Instrumentation
some samples of carrier ampholyt’es inhibit the riboflavin-cat,alyzed react.ion. Some protein samples are also inhibitory. I t is then preferable to use an alternative procedure in which persulfate-cabalyzed polymerization is allowed to proceed with the carrier ampholytes incorporated into the solut,ion, and to add the sample snbeequently on top of the gel, as is done for disc electrophoresis ( 1 4 ) . Contact between persulfate and t,he sample is avoided by a short) preliminary focusing. If this is done with the cathode at the top and the anode at the bottom, it is not’ necessary for all of the persulfate to be out of the gel before the sample is applied, because any remaining persulfate \vi11 move don-n ahead of the prot.ein when the current is turned on. After the preliminary focusing, the sample, in a solution of ampholytes and 10% sucrose, is carefully introduced above the gel under a layer of ampholytes in 5% sucrose which separates it, from the electrode solution. The resolution obtainable wit,h gel electrofocusing may be illustrat,ed by describing an experiment carried out with crystnlline L-amino acid oxidase. an enzyme purified from the venom
Figure 2. Isozymes of L-amino acid oxidase separated by gel electrofocusing (left) and by gel electrophoresis (right) (from ref 10). For experimental details, see text
of the Eastern Dia,mondback rattlesnake (Crotalus adamanteus) (10). The result, of this experiment) is shown in Figure 2. For comparison, n disc electrophoresis experiment performed wit,li the same sample of enzyme is also shown. The gel used in the electrofocusing experiment, was made by photopolymerizntion of a solution containing the following component,s: acrylamide, 7.5% : N,N’-met’hylene bisacrylnmide, 0.2% ; lf7::Z’,h”,AV’-tetramethylethylenediamine, 0.058% : carrier ampholytes (“Ampholine,” p H 3-10), 2% : enzyme, about 50 pg/ml: and riboflavin, 0.0015%. After deaerating about 3 ml of this solution by placing it under reduced pressure for 1 to 2 min. it was pipetted into a glass tube, G x 120 mm, closed off a t the bottom nit11 Parafilm. The solution r a s covcred ~ i t ha 1-em la!.er of water and allowed to gel at room temperature for 30 min. Electrofocusing was carried out at 4’ n i t h a final voltage of 120 1- for 9 lir in the apparatus slionn in Figure 1. The anode (top) was immersed in 1 9 H,SO, and the cathode in 2.9% ctlianolamine. To prevent overhenting, the voltage was increased gradually to its final value, the power outp i i t aln-ay. being kept below 0.2 R per gel. The optimum focusing time and final voltnge used depend on a number of fnctors. such as the length of the pel, the pH range of the carrier ampholytee, and the concentration of salts or buffer initially present in the sample. TS’hen electrofocusing is complete, the gel is removed from the tube. I t ma!- then be fixed and stained for protein. Alternatively it may be stained for a specific enzymatic activity or analyzed in a variety of other ways. Because amido black and some other protein stains form complexes wit,li the carrier ampholytes, the>- do not give satisfactory results unless the fixed gel is first extensively n-ashed. Such wvasliing is not necessary, however, ~vhen the protein is stained with Coomnsaie blue. A method b y which tlic gel may be simultaneously fixed and stained is to immerse it overnight in a solution containing 5% trichloroacetic acid, 5% sulfosalicylic acid, 18% methanol, and 0.02% Coomassie blue. Excess stain is then removed by washing the gel in water. -1 convenient method of verifying that a pH gradient has been established and of measuring the pH in various regions of the gel is to touch t8hesurface of the gel at various points n.itli a microelectrode (combination glass and reference electrode, Cat. No. 14153, available from Instrumentation Laboratory, Inc., Watertown, Mass.).
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ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
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ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
This slioiild be done immediately after removing the gel from the tube. The gel is laid alongside a ruler and touched at 1-em intervals with the electrode in sucli :Lway that both the glass membrane and the reference junction are in contact with the surface. These meahiirenients take leas than 5 min per gel. Comparison of Gel Electrofocusing and Gel Electrophoresis
-4s shown in Figure 2 , gel electrofocusing revealed the presence of at lea.st, 18 protein components in cryatalline L-amino acid ositlnse, whereas only three zones could be separated by gel electropliorcsis. It could be shoir-n, by comparing tlie gels stained for protein with Coomassie blue n-ith similar gels stained specifically for L-amino acid ositlne activit!. using a stnining solut,ion containing L-leucine, phenazine nicthosulfate. and nitroblue tetrazoliiim, that all of tlie protein bands enzyinntic activity. The - nns considered that these uld rcpresent nn artifact arising from nn interaction between the enzyme a n d the gel or from an alteration of the enzyme in the coiirse of purification. However, sucli artifacts were ruled out by showing that, a similar pnttern of zones TKIS obtained n-11~11electrofociising was conducted in n iiicrose density gradient or when unt rented m i k e w n o m was electrofocused in gels niid stained for enzymatic activity ( I O ) . I t mny therefore be concluded that tlicre are at least 18 isozymes of Lamino acid ositlnse. perhaps n larger number than has been found for any other cnz!’me. This raises many interesting questions concerning the nature of the chemical differences between the isozymes. their origin, and t,Iicir physiological significance. It
