Apparatus for the electro-elution and collecting of electrically charged macromolecules in a trap

In an electro-elution apparatus for operation in an electrophoresis chamber and for the electro-elution of biological macromolecules from sections of electrophoresis gel, the macromolecules which are to be eluted are not temporarily adsorbed in a gel, but can be collected between two polymer membranes in a trap and pipetted out of the latter. The membrane, which is the first or inner membrane in the direction of migration of the macromolecules, is here permeable to the macromolecules under the action of the electric field, while the membrane, which is the second membrane in the direction of migration, namely the outer membrane of the electro-elution apparatus, is impermeable to the macromolecules under the same conditions. After the electric field has been switched off, both membranes are at least substantially impermeable to any mass transfer.

DESCRIPTION 
The invention relates to an apparatus for the electro-elution of 
electrically charged macromolecules. 
Specifically, the invention relates to a process for the electro-elution of 
biological macromolecules, which as a rule are electrically charged, from 
electrophoresis gels. 
An apparatus of this type has been disclosed in the journal Analytical 
Biochemistry 124, 299 to 302 (1982). The known apparatus is a plate-shaped 
cuboid of polyacrylate glass, in which several mutually parallel elution 
channels are formed in the shape of U-grooves which extend continuously 
from one side of the cuboid up to the opposite side and are open at the 
top. In one end region of each of the elution channels, nylon gauze in the 
form of a bag is inserted, which is hermetically sealed in the manner of a 
filter from the channel walls and is filled with an adsorption gel for the 
macromolecules, for example DNA, RNA, proteins or lipopolysaccharides. 
The problem of electro-elution arises when the electrophoresis gels, in 
which macromolecules are usually fractionated, are worked up. The piece of 
electrophoresis gel containing the target fraction is cut out and placed 
into the channel of the electro-elution apparatus. The apparatus is then 
placed into a horizontal electrophoresis chamber in such a way that the 
electric field, which can be generated in the electrophoresis chamber, is 
parallel to the elution channel of the electro-elution apparatus. The 
electrophoresis chamber is then filled with a buffer to a level just below 
that at which there is a flow over the separating walls located between 
the mutually parallel channels. When the electric field is switched on, 
the electrically charged biological macromolecules migrate out of the 
electrophoresis gel lying in the buffer and are collected in the 
adsorption gel placed in the nylon bag. When the elution is complete, the 
elution gel is removed from the nylon bags and eluted on a column out of 
this adsorption gel and, in particular when the conventional malachite 
green gel is used, as a rule by means of a one-molar sodium perchlorate 
solution. 
The complete procedure for this process, which at present is the best known 
to the state of the art, requires approximately forty minutes per piece of 
electrophoresis gel and, due to the repeated adsorption and elution, 
entails significant losses of target substance. Even if bacterial losses 
are disregarded, a total loss of target substance of at least about 25% 
must be expected in this elution process, that is to say at best a yield 
of approximately 75%, relative to the weight of the macromolecules in the 
initially introduced piece of electrophoresis gel, can thus be expected. 
In addition, the costs of the temporary adsorption gel are considerable. 
Thus, the commercial price for 25 ml of malachite green gel is, for 
example, about DM 350.00. 
In the light of this state of the art, it is the object of the invention to 
provide an apparatus for the electro-elution of electrically charged 
macromolecules, in particular biological macromolecules, which permits 
rapid, loss-free and inexpensive elution of macromolecules, in particular 
from electrophoresis gels. In the sense of this object and in the sense of 
the present invention, the terms "elution" or "electro-elution" are to be 
understood also as the "elution" of the macromolecules from liquid phases 
under the action of an electric field, that is to say in particular the 
concentrating of solutions, desalination or transfer into a different 
buffer. 
To achieve this object, the invention provides an apparatus for 
electro-elution, wherein, the trap for the target macromolecules is 
bounded or formed by two polymer membranes. Of these two membranes, that 
which is the outer one relative to the longitudinal axis of the elution 
channel is permeable in the presence of an electric field to small ions 
and molecules, in particular to buffer and water, but is impermeable to 
the macromolecules which are to be eluted. That membrane which is the 
inner one in this sense, however, is permeable to all ions and molecules, 
that is to say in particular also to the macromolecules to be eluted, if 
this membrane is located in an electric field. With the electric field 
switched off, however, both membranes are impermeable to any ions and 
molecules of whatever size, that is to say, in particular, they are 
virtually water-tight. With the electro-elution medium made up and the 
electric field switched on, the macromolecules are thus extracted, for 
example, from the initially introduced piece of electrophoresis gel, 
migrate through the inner membrane in the direction of the electric field, 
as a rule towards the positive pole, and are retained and collected, that 
is to say concentrated, on the inside of the outer membrane, whereas the 
buffer ions migrate through this membrane into the electrophoresis 
chamber. 
For operation, the apparatus according to the invention is placed into the 
electrophoresis chamber in the same way as the known apparatus described 
above. After the elution process has been completed, the polarity of the 
electric field is briefly reversed, preferably for ten to fifteen seconds, 
so that the macromolecules collected, and sometimes also partially 
adsorbed, on the inner surface of the outer membrane are detached from the 
membrane surface and released into the trap space. 
With this apparatus, the electro-elution of a piece of electrophoresis gel 
takes not more than five minutes. The yield of eluted macromolecules is 
here at least 90% and can be virtually 100%. This yield can be obtained 
because the inner membrane used is a membrane which is water-tight and 
thus additionally bacterial-tight, that is to say it does not allow any 
bacteria to be transferred from the elution channel or the elution chamber 
into the trap. If the trap was carefully sterilized beforehand, a loss of 
macromolecules due to bacterial attack, which can take place to a 
considerable extent in all known electro-elution processes, can be 
eliminated in this way. 
A great variety of membranes which meet the requirements stated above are 
commercially available in the most diverse shapes. Preferably, polymer 
membranes are used, the structure of which is based on polymers which can 
be wetted by water and the buffer solutions used, such as, in particular, 
cellulose, cellulose derivatives, polyamides, polyimides and polysulfones. 
Preferably, membranes of cellulose acetates are used as the outer 
membranes and those of regenerated cellulose are used as the inner 
membranes. Such membranes are available, for example, from the assignee of 
this application, under the type descriptions RAB or RAC for the outer 
membrane and RSB for the inner membrane. A large selection of the 
cellulose membranes to be used here can, however, also be obtained from 
any other manufacturer. The inner membrane should here have a mean pore 
size in the region of about .ltorsim.0.2 .mu.m, in particular a pore size 
in the range from 0.05 to 0.20 .mu.m, and the outer membrane should be 
impermeable to macromolecules having a molecular weight greater than 
approximately 1,000. It is, however, self-evident that such data are to be 
regarded only as guide values which can and will readily be changed by the 
user in accordance with the particular problem to be solved. 
According to an embodiment of the invention, the macromolecule traps formed 
by the particular inner membrane and the particular outer membrane are 
provided on the two end regions of the continuously open elution channel, 
so that a separate elution chamber is formed between the two mutually 
opposite inner membranes in each case. This enables a buffer solution 
different from that in the electrophoresis chamber, in which the apparatus 
according to the invention is operated, to be used in the elution chamber. 
This makes it possible to desalinate the macromolecule solution or to 
change the buffer. 
The membranes are preferably mounted exchangeably in the body of the 
apparatus. The exchangeability can here be obtained in particular by 
inserting or pushing the membrane from above into corresponding guides or 
recesses in the apparatus body. Above all for the membranes located on the 
inside, it has here proved to be convenient, handy and 100% reliable, if 
the inner membrane is surrounded by a frame of swellable material and this 
frame, in the dry and unswollen state, can be pushed with an exact fit 
into a U-shaped slotted guide open at the top. After the elution chamber 
has been filled with elution medium, or the apparatus has been placed into 
the horizontal electrophoresis chamber, the elution medium flows around 
the frame and causes it to swell, so that the inner membrane is clamped 
like a filter, making a seal, into the channel walls. 
The outer membrane, which can also be used unframed, preferably bears from 
the outside against a separating wall which bounds the trap axially 
outwards. In this solid separating wall, a central bore which is covered 
by the membrane is provided for the passage of the ion-current. The 
membrane is here clamped or pressed against the separating wall and over 
the hole by a frame which in turn is stressed axially inwards, relative to 
the channel of the apparatus, for example by means of a compression spring 
or a tensioning screw or tensioning-screw sleeve. The seal between the 
outer membrane and this separating wall can additionally be improved by 
one or more sealing rings or sealing edges. Preferably, a continuous 
channel, which opens conically outwards, is here formed in the frame and 
in the tensioning-screw sleeve in order to avoid the formation of gas 
bubbles. 
The body of the apparatus according to the invention is preferably made 
from an inert plastic, for example from a polycarbonate or an acrylic 
glass. Preferably, the plastic is suitable for autoclaving.

FIG. 1 shows the plan view of an illustrative embodiment of the 
electro-elution apparatus according to the invention or, more precisely, 
the body 1 of such an apparatus. FIG. 2 shows an axial section of this 
illustrative embodiment, the outer membrane M1 and the inner membrane M2 
having been inserted on the left in the representation of FIG. 2. The body 
1 consists of transparent polycarbonate. The membrane M1 is a cellulose 
acetate membrane which is impermeable to molecules having a molecular 
weight of greater than 1,000, even in the presence of an electric field. 
The inner membrane M2 is a simple cellulose membrane of a pore size of 0.2 
.mu.m. The space defined and bounded between the membranes M1 and M2 
serves as the trap 2 for the macromolecules which are to be eluted. 
Axially outwards, the trap 2 is bounded by a separating wall 3 which is 
formed integrally from the body 1. In the separating wall 3, a relatively 
large orifice 4 is provided which ensures a free connection between the 
trap 2 and the surroundings. Specifically, the orifice 4 opens into a 
recess or prechamber 5 which is cut out with an open top and is axially 
open towards the end face of the body 1 via a bore which has an internal 
thread 6. With the apparatus in operation, this prechamber 5 is preferably 
filled with air. For this purpose, the a tensioning sleeve 7 with an 
external thread must be formed in such a way that, after screwing into the 
internal thread 6, the system is water-proof outwards, that is to say into 
the prechamber 5. A tensioning frame 8 which is guided, secure against 
rotation, on the side walls 9, 10 of the prechamber 5 is, axially inwards, 
inserted into and pushed over the tensioning sleeve 7. Around its 
circumference, the tensioning frame 8 has a sealing and cutting edge, the 
clear diameter of which is somewhat greater than the diameter of the 
orifice 4. When the tensioning sleeve 7, with the tensioning frame 8 
inserted, is screwed into the body 1, the tensioning frame 8 is guided 
towards the separating wall 3. At the same time, the membrane M1 inserted 
from above and the orifice 4 are clamped in between the tensioning frame 8 
and the separating wall 3, making a tight seal. By means of the sealing 
and cutting edge 21, the membrane M1 itself is here used as a gasket. 
An orifice 12, aligned with the orifice 4, in the tensioning frame 8 
ensures free flow of current through the membrane M1. The orifice 12 in 
the tensioning frame and the neighbouring orifice 21 in the tensioning 
sleeve form a cylindrical channel (FIG. 2) or, preferably, widen conically 
outwards (FIG. 5) and thus prevent the persistence of gas bubbles in front 
of the membrane M1. The tensioning sleeve 7 with an external thread can be 
screwed into the internal thread 6, and the tensioning frame 8 which is 
guided, secure against rotation, on the side walls 9, 10 (FIG. 1) of the 
prechamber 5 is axially inwards inserted telescopically into and pushed 
over the tensioning sleeve. By screwing the tensioning sleeve 7 in, the 
tensioning frame 8 is guided towards the separating wall 3 or forced onto 
it. At the same time, the outer membrane M1 insertable from above into the 
prechamber 5 is clamped in, firmly and tightly sealing the orifice 4, 
between the tensioning frame 8 and the separating wall 3. The tightness of 
this clamping can be improved by additional sealing rings 11. The orifice 
12, aligned with the orifice 4, in the tensioning frame 8 ensures a free 
flow of current through the membrane M1. Since the prechamber 5 is kept 
free of liquid, the tightness of the trap 2 towards the prechamber 5 can 
be monitored in an optimum manner, and the formation of tracking current 
paths can be excluded. 
Axially inwards, the boundary of the trap 2 is fixed by a U-shaped slotted 
groove 13 which is open at the top and into which the inner membrane can 
be inserted from above. The U-shaped slotted groove 13 is here formed both 
in the side walls 14, 15 and in the floor 16 of the channel 17. 
The membrane M2 is provided with a frame-like edge zone which, in the dry 
state, can be inserted with an exact fit into the slotted groove 13. When 
the elution medium is introduced into the channel 17, the frame of the 
membrane M2 swells, so that the membrane is retained in the slotted groove 
13 with a hermetically sealed press fit. 
Between the two inner membranes M2, a section, serving as the elution 
chamber 18, of the continuous channel 17 is separated off by these 
membranes. On the one hand, the elution chamber 18 is electrically 
connected to the outer electrolyte of the electrophoresis chamber via the 
membranes M1 and M2 and the orifices 4 and 12 but, on the other hand, it 
is hydrodynamically and chemically separated from the surroundings to such 
an extent that it is quite possible to fill the elution chamber 18 with an 
electrolyte or buffer of a composition which is substantially different 
from that of the electrolyte used in the electrophoresis chamber. 
For operation, the gel piece which is to be eluted and which contains the 
macromolecules, is placed into the elution chamber 18 of the 
electro-elution apparatus. The two traps 2 and the elution chamber 18 are 
then filled with a highly dilute buffer or with distilled water, namely in 
such a way that the level 19 (FIG. 3) of the medium filled in is just 
below the upper edge 20 (FIG. 2) of the chamber 18 or the body 1. The 
apparatus is then placed into a horizontal electrophoresis chamber which 
is filled with so much buffer solution that the level of the buffer 
solution in the electrophoresis chamber is at least essentially the same 
as the level 19 in the elution chamber. However, the concentration of the 
buffer solution in the electrophoresis chamber can here be substantially 
greater, as a rule ten to a hundred times greater, than the concentration 
of the buffer in the elution medium. 
Moreover, the electro-elution apparatus is placed into the electrophoresis 
chamber in such a way that the electric field, which can be generated in 
the electrophoresis chamber, is at least substantially parallel to the 
channel 10. This is shown diagrammatically in FIG. 3. In the illustration 
in FIG. 3, the minus signs surrounded by a large circle here denote 
negatively charged macromolecules, whilst the plus signs and minus signs 
surrounded by a small circle denote the buffer ions. In the presence of 
the electric field, the polarity of which is assumed to be as shown in 
FIG. 3, the ions migrate in the directions shown in FIG. 3 by short 
arrowheads, namely the positive buffer ions migrate to the negative pole, 
and the negative buffer ions and the negatively charged macromolecules 
migrate to the positive pole of the acting electric field. At the same 
time, all the negatively charged particles pass through the membrane M2 
into the trap 2. However, whilst the negatively charged buffer ions then 
also pass through the outer membrane M1 under the action of the field and 
are thus transferred into the buffer of the electrophoresis chamber, the 
electrically charged macromolecules are retained on the axially inward 
surface of the outer membrane M1. 
In this way, all the electrically charged macromolecules are gradually 
collected on the membrane M1. After the elution has been completed and all 
the electrically charged macromolecules have been transferred from the 
elution chamber 18 into the trap 2, the polarity of the field is reversed 
for about ten to fifteen seconds, so that the macromolecules collected on 
the outer membrane M1 are released and migrate into the trap 2, from where 
they can readily be removed in high concentration from above, for example 
by means of a micropipette. 
The volume ratio of each individual trap 2 and the elution chamber 18 is 
approximately 1:100. However, since the trap 2 must not be made so narrow 
that it is no longer accessible, for example, to a pipette, the volume 
reduction is effected in the manner shown in FIG. 2 by raising the floor 
or bottom of the trap 2 relative to the floor of the elution chamber 18. 
Alternatively, the volume of the trap can be reduced by the methods evident 
from FIGS. 4 and 5, namely by making the trap 2 with a circular 
cross-section and a downward conical taper. 
The further illustrative embodiment of the electro-elution apparatus, as 
shown in FIGS. 4 and 5, differs additionally from the apparatus shown in 
FIGS. 1 and 2 in that the channel 21, defined by the orifice 12 in the 
tensioning frame 8 and the interior of the tensioning sleeve 7, is made 
continuous and with outward-opening smooth walls. This prevents the 
formation or adhesion of gas bubbles in the channel 21 and hence changes 
in the electric current conditions. 
Moreover, in the tensioning frame 8 shown in FIG. 5, a cutting ring edge 22 
is formed, which forces the membrane M3 against the outside of the 
separating wall 3, making a seal.