Note: Descriptions are shown in the official language in which they were submitted.
2032~87
S011355.16
"Process and apparatus ~ treating
electrophoresis carriers"
The invention relates to a process and an apparatus
for treating electrophoresis carriers. More
particularly it relates to a process and apparatus in
which the liquid used for treatment flows along the
electrophoresis carrier which is to be treated.
The analysis, determination or characterisation of
colloidal and macromolecular substances, e.g. proteins,
is simply and expediently carried out with the aid of
electrophoresis. This well-known method of measurement
comprises the steps of electrophoretic separation and
after-treatment, by means of which the fractions of the
sample are fixed on the carrier and prepared for
subsequent evaluation. If the fractions are to be
analysed or measured by optical methods, the after-
treatment serves to make the fractions visible on the
electrophoresis carrier. For this purpose a number of
chemical reactions (e.g. staining and decolorising) are
required. These reactions or procedures are separated
from one another by washes. A number of electrophoresis
carriers have to be washed out before being used in
electrophoresis (in order to remove unwanted ingredients
(e.g. monomers remaining from the polymerization)) and
possibly have to be charged with water, buffer solution
or reagent (e.g. urea).
Conventional electrophoresis carriers are gels
(e.g. agarose gel, polyacrylamide gel). These are used
either with solid carriers or with no additional
carriers. The gels may for example be polymerised on
films or poured onto sheets (for horizontal
electrophoresis) or poured between two sheets (for
vertical electrophoresis). It is also possible to use
gels reinforced with netting. The electrophoresis
carriers used or currently available are generally of
2032~87
-- 2
the dimensions, e.g. (in cm): 30 x 20, 16 x 18, 3 x 8,
10 x 15, 7 x 15, 5 x 1 or 5 x 2. These electrophoresis
carriers are used in all sorts of thicknesses in
conventional methods. Cylindrically shaped
electrophoresis carriers are also still in use. The
carriers mentioned can in most cases be used for both
one-dimensional and two-dimensional electrophoresis.
US Patent No. 4391689 describes an apparatus having
a large number of tanks provided for the individual
steps of the method of measurement. In this apparatus
the electrophoresis carrier is mechanically conveyed
from one tank to another. US Patent No. 4222843
describes an apparatus in which there is a drum which
carries the electrophoresis carrier and repeatedly dips
it into a tank provided underneath. The liquids needed
for the reactions and washes are placed one after
another in the tank. W087/05111 describes an apparatus
for the after-treatment of electrophoresis carriers
which consists of a container having on its lid a
rotatable retaining device in which the electrophoresis
carrier is clamped. This retaining device moves the gel
in the tank and hence in the liquid provided.
All these apparatus imitate the old method which is
still most frequently used, in which the electrophoresis
carrier is manually moved from one liquid container to
another for the individual steps of the after-treatment
and agitated therein either manually or on a vibrating
device. In the apparatus mentioned the batch process is
used. The liquids are supplied discontinuously or the
electrophoresis carrier is introduced, for each step,
into the particular liquid required.
We have now been able to provide a process and
suitable apparatus by means of which the treatment and
more particularly the after-treatment or development of
the electrophoresis carriers can be carried out easily,
in a labour-saving manner and, if desired,
automatically.
`` 2032~,~7
-- 3 --
25771-570
The lnvention generally re~ates to a process for treat-
ing electrophoresis carriers in which a liquid used for treatment
is caused to flow around and over the electrophoresis carrier in
contact with it.
The invention further relates to an apparatus in which
electrophoresis carriers may be treated comprising a container
with, in spaced relationship, inlet and outlet means for a liquid
used in the treatment, in which in use the liquid flows through
at least part of the container between the inlet and outlet
means~ and the electrophoresis carrier which is to be treated is
located during treatment in that part of the container through
which the liquid flows.
As used herein, the expression "container" is intended
to include conduit or other means in which the carriers may be
situated whilst treatment liquid flows.
If the treatment or after-treatment of the electro-
phoresis carrier comprises a number of steps, the respective
liquids may be passed successively through the container. The
process and apparatus according to the invention may be used both
for the treatment of electrophoresis carriers before electro-
phoresis (e.g. for the above-mentioned washing out and charging)
and for the after-treatment or development (e.g. staining) of
electrophoresis carriers on which the fractions of the separated
samples are located.
In the process according to the invention for treating
electrophoresis carriers, the carrier stays in the same container
during all the treatment steps. The particular liquid required
is introduced into the container and allowed to flow way. If
- 3a - 2032~8~
25771-570
the treatment step is a longer-lasting one, the liquid ls
passed continuously through the container for a suitably long
period. The liquid is therefore allowed to or caused to flow
around the electrophoresis carrier. The chemical reaction or
washing process is thus made more intensive, since the flow
action means that the liquid in contact with the surface of the
electrophoresis carrier is constantly renewed. There is no need
for mechanical movement of the electrophoresis carrier. The
liquid is
- 2~32887
either constantly renewed or circulated.
The process according to the invention and the
apparatus suitable for performing it are suita~le for
all the above mentioned electrophoresis carriers. It is
advisable to keep smaller pieces in frames either
individually or in groups.
The apparatus according to the invention for
treating electrophoresis carriers or for the after-
treatment of electrophoresis carriers on which the
fractions of the samples being analysed are located has
a container with inlet and outlet means for the liquids
used for the treatment. The inlet and outlet means are
located in various areas of the container, for example
they may be provided at opposite ends to each ather.
The liquids flow freely through the volume of the
container between the inlet and outlet means. During
the treatment the electrophoresis carrier is situated in
this part of the container through which the liquid
flows. By regulating the pressure at which the liquid
is introduced into the container it is possible to
regulate the intensity of flow of the liquid. The
apparatus is preferably constructed so that the
direction of flow obtained is either substantially
vertical or horizontal. By suitable arrangement of the
inlet and outlet means in the container it is possible
to achieve a direction of flow which runs between the
vertical and horizontal. In a preferred arrangement a
substantially vertical direction of flow is generated,
the flow being directed upwards. If, for example, an
SDS gel (polyacrylamide gel with added SDS [sodium
dodecyl sulphate]) with no fixed carrier is introduced
into an apparatus of this kind, the gel may be kept
suspended in the liquid by the flow of liquid. If the
flow rate is great enough, the electrophoresis carrier
is aligned in such a way that the large areas of its
surface are substantially parallel to the direction of
flow of the liquid.
2032887
The electrophoresis carriers may also be retained
in a specific position in the container by conventional
retaining means. These retaining means grip the
electrophoresis carrier at the top and/or bottom and/or
sid~s. The retaining means may also be in the form of a
frame. Such a frame may be constructed, for example, so
as to contain one chamber or a plurality of chambers the
walls of which are formed by a small-meshed grid. If
the electrophoresis carriers are retained by such means
or frames, these retaining means/frames are preferably
held parallel to the direction of flow in the apparatus.
If retaining means/frames are used it is also possible
for treatment liquid to flow vertically from top to
bottom.
The process, apparatus and function of the
apparatus will now be more particularly described by
means of the Examples and accompanying drawings which
follow, which are by way of Example only.
Fig. 1 shows a simple embodiment of an apparatus
according to the invention for treating an
electrophoresis carrier. This apparatus consists of a
cylindrical container (1) provided with an inlet tube
(2) and an outlet tube (4). The inlet tube (2) opens
into the annular duct (3). A grid (5) may be suspended
in the container (1). The grid (5) covers the entire
opening of the container (1). The inlet tube and outlet
tube are provided with the necessary regulating element
(not shown). The outlet tube (4) is provided, at its
end projecting into the container, with a sieve. The
annular duct consists of a pipe running concentrically
with the inner wall of the container. The upper half of
the wall of the pipe is provided with openings (6). The
liquid for each treatment stage is supplied under
pressure through the inlet tube (2), passes through the
openings in the annular duct into the container and
flows out through the outlet tube (4). Since the liquid
passes under pressure through the openings in the
~3~g87
annular duct into the container, a flow is formed. The
pressure of the liquid is adjusted so that the
electrophoresis carrier placed therein is kept suspended
in the liquid without reaching the grid (5). I'he grid
(5) serves only to hold the electrophoresis carrier back
and prevent it reaching the outlet tube should the flow
become too strong as a result of an operational error.
Fig. 2 shows a cross section through the annular
duct (3). An opening (6) is a hole drilled through the
wall of the tube extending radially at an angle of 45
to the base of the container.
Fig. 3 shows a plan view of the inlet tube (2) and
the annular duct (3). The openings (6) are arranged in
a circle at the same height above the base of the
container. The circle is located on that side of the
tube of the annular duct which faces the centre of the
container.
The size of the container is selected so that the
electrophoresis carrier has just enough room (without
being bent or kinked) in the part of the container
through which the liquid flows. If the apparatus
according to Figs. 1 to 3 is to be used, for example,
for treating a piece of SDS electrophoresis gel
measuring 7 x 15cm or up to 10 x 15cm, it is advisable
to use a container with approximately the following
dimenslons:
Internal diameter of container (1): 180 mm
Internal height of the container (1): 240 mm
Spacing of the outlet tube (4) above
the base of the container:180 mm
Spacing of the annular duct (3) above
the base of the container:2 mm
External diameter of the annular
duct (3): 120 mm
External diameter of the tube of
the annular duct (3): 10 mm
2032887
Internal width of the openings in
the annular duct (3): 1 mm
Number of openings in the annular
duct: 10
The liquid is introduced under a pressure of at least 60
millibars. The electrophoresis gel is kept afloat in
the liquid without being washed up against the grid (5).
Fig. 4 shows an alternative embodiment of the
apparatus according to the invention. This co~prises a
cylindrical container (7), which has a conical,
downwardly extending base with an opening (10) and tube
connection for the inlet and outlet means. An inlet
tube (8) with an annular duct and an outlet tube (9)
correspond to the analogous components in the apparatus
according to Fig. 1. The cylindrical part of the
container is separated from the conical part of the
container by a protective grid (11). This protective
grid prevents the electrophoresis gel introduced into
the container from obstructing the opening (10~. The
inlet and outlet tubes are fitted with the necessary
regulating means (not shown).
In this alternative embodiment of the apparatus,
the liquid can be drained rapidly and completely away
from the container when desired through the opening
(10). For a short treatment step or for washing the
container, the liquid in question can be drained away
through the opening (10) and possibly fed in again.
`` 2032~87
-- 8
Example 1
By way of an example of the use of the apparatus
according to the invention, the Coomassie-blue-staining
of an SDS electrophoresis gel (polyacrylamide gel with
SDS (sodium dodecyl sulphate) added thereto) will now be
described, using the embodiment of the apparatus shown
in Fig. 4:
After the end of electrophoretic separation the gel
is placed without a carrier plate in the container (7).
The staining solution is supplied under pressure through
the inlet tube (8), drained away through the outlet tube
(9) and circulated by pumping round in this way for 20
to 30 minutes. The flow of liquid thus produced keeps
the gel floating in the liquid. The container is then
totally emptied through the opening (10). Then the
decolorizing solution is introduced into the container
through the inlet tube (8) for 30 minutes under pressure
and drained away through the outlet tube (9). Fresh
decolorizing solution is supplied constantly. (It is
also possible to recycle the decolorizing solution by
including a regeneration stage.) The flow of the liquid
causes the gel to remain afloat in the liquid. This
process (decoloriæing) is continued until the substrate
is (approximately) colourless. Then the decolorizing
solution is totally drained away through the opening
(10). Then a 2% aqueous glycerol solution is introduced
through the inlet tube (8), drawn off through the outlet
tube (9) and pumped round for 20 minutes in this way.
Then the gel is taken from the container and dried.
Example 2
As a further example, the silver staining of an SDS
electrophoresis gel (polyacrylamide gel with added SDS
(sodium dodecyl sulphate)) according to Oakley will now
be described using the apparatus according to Fig. 4.
After the electrophoretic separation has ended, the
gel without a carrier plate is placed in the container
2~32887
g
(7).
Step 1
The decolorizing solutionl used to remove the
bromothymol blue (for characterising the front) is
supplied under pressure through the inlet tube (8),
drawn off through the outlet tube (9) and recycled. The
flow of liquid thus produced causes the gel to stay
afloat in the liquid. This decolorization is carried
out for 20 minutes. In order to remove all the
decolorizing solution it is drained away through the
opening (10).
Step 2
The container (7) is then filled with deionized
water and emptied again immediately. The water is
supplied and removed through the opening (10).
Step 3
10% glutardialdehyde solution is supplied under
pressure through the inlet tube (8) and drawn off
through the outlet tube (9). The gel floats in the
liquid. This treatment is carried out for 20 minutes,
with the solution being recycled constantly by pumping.
In order to drain it away completely the
glutardialdehyde solution is drained away through the
opening (10).
Step 4
Washing with deionized water is carried out
analogously to step 2. This washing is carried out at
least 3 times.
Step 5
The gel is intensively rinsed with deionized water
for 30 minutes by introducing the water under pressure
through the inlet tube (8) and draining it away through
-- 2032~87
-- 10 --
the outlet tube (9). Fresh deionized water is
constantly supplied. Then the water is drained away
completely through the opening (10).
Step 6
Rinsing as in step 2.
Step 7
Ammoniacal silver nitrate solution is introduced
into the container through the opening (10) until the
gel is covered. The arrangement is left to stand like
this for 15 minutes.
It is also possible to perform this step by
supplying the silver nitrate solution under pressure
through the inlet tube (8) and draining it away through
the outlet tube (9). The silver nitrate solution is
recycled for 10 minutes.
After this treatment, in both alternative
embodiments, the silver nitrate solution is drained away
completely through the opening (10). The container is
briefly cleaned 3 to 5 times by supplying deionized
water through the opening (10) and then letting it run
away. If the tubes (8) and ~9) have been used, these
must also be thoroughly cleaned.
Step 8
The developer solution is supplied through the
inlet tube (8), drained away through the outlet tube (9)
and recycled. After 5 to 10 minutes the developer
solution is drained away through the opening (10). (The
retention time of the developer solution must be chosen
to suit the particular sample.)
Step 9
For half an hour, deionized water is supplied under
pressure through the inlet tube (8) and drawn off
through the outlet tube (9). Fresh water is supplied
2032887
-- 11 --
constantly.
Other after-treatment steps may be added on in the
same way. When the after-treatment has ended the gel is
taken from the container and prepared for storage.
Example 3
Washing of a polyacrylamide gel which is,
optionally, dried after the washing process has ended,
thus becoming suitable for storage and rehydration:
Step l
After the polymerisation has ended, the
polacrylamide gel (optionally a fabric-reinforced gel)
is placed in the container filled with deionised water
(according to Fig. 4).
Then fresh deionised water is allowed to flow from
the bottom to the top of the container for 30 minutes so
that the gel remains suspended or afloat. In so doing,
all the dissolved substances, preferably monomer
acrylamide, monomer cross-linking agents and excess
ammonium persulfate are washed away.
Step 2
After the washing has been carried out, the supply
of demineralised water is stopped and the washings are
removed through the opening in the bottom.
After the container has been completely emptied, it
is filled with an aqueous glycerol solution and this
solution is circulated by pumping round for 15 to 30
minutes. Then the solution is drawn off through the
opening. Shortly before the solution is completely
removed, the gel is taken from the container.
SteP 3
The gel is placed onto a hydrophobic base, covered
with a film of polyester and by slightly pressing it
down uniformly the excess liquid is pressed out. Then,
2032g~7
- 12 -
usin~ conventional methods (drying by air or microwave)
the gel is dried. Gels thus treated are suitable to
rehydration, even after long storage.
Polyacrylamide gels set on a carrier may also be
treated analogously.
The Examples may be carried out analogously using
other electrophoresis carriers which may optionally be
held in a frame, as described hereinbefore, or other
retaining means.
The apparatus according to the invention may be
modified in various respects.
The inlet means, in the form of an annular duct, as
described in the above examples may also be designed in
other forms known per se. An annular duct which is
fixedly connected to the casing of the container may be
provided. The annular duct with the outlet openings for
the inflow of liquid may also be replaced by a sieve
plate. In small containers, in particular, the annular
duct may be replaced by a straight tube having the
corresponding openings. Instead of such a tube, a slot-
like opening may be provided in the wall of the
container for the inflow and possibly for the outflow.
This arrangement is also particularly advantageous if an
electrophoresis carrier is treated in the horizontal
position. The apparatus can then be constructed as
follows, for example: the direction of flow of the
liquid is horizontal, the container is of low height,
the slots for the inlet and outlet of liquid are
provided on opposite sides of the container,
substantially on an extension of the longitudinal
surface of the electrophoresis carrier.
The base of the container may be flat or domed.
If a plurality of electrophoresis carriers are to
be treated simultaneously in one container, it is
advisable to distribute the inlet and outlet of the
liquid uniformly over that part of the container through
which the liquid flows. This may be achieved for
- 2032887
- 13 -
example by providing a plurality of annular duats, inlet
tubes or slots side by side (the number preferably
corresponding to the number of electrophoresis carriers
to be treated).
The size of the apparatus is advantageously
selected in accordance with the size of the
electrophoresis carriers used. As a guide for the size
of the container, the individual electrophoresis carrier
should be surrounded on all sides by a layer of liquid
at least l to 10 mm deep, preferably at least 3 to 5 mm
deep.
If electrophoresis carriers of greatly differing
sizes are to be treated in the same apparatus, it is
advisable to construct the outlet ((4) in Fig. l, (9) in
Fig 4.) so that the height of the level of liquid can
vary with the size of the electrophoresis carrier. This
can be achieved, for example, by providing outflow
openings at different heights in the container. For
treatment of a small electrophoresis carrier, for
example, one of the lower outlet openings is used.
Another possibility is to design the outflow opening to
be movable over part of the height of the container
wall.
For using the above mentioned frames, corresponding
retaining devices are provided, e.g. rails into which
the frames can be inserted.
The container of the apparatus may also be
surrounded by heating coils/heat exchangers. The liquid
may also be introduced into the container at the
required temperature. Consequently, each treatment step
can be carried out at the optimum constant temperature.
Moreover, the results for series of tests can be
standardised, thanks to the constant temperatures of the
treatment. If the treatment is carried out at elevated
temperature, the treatment time is reduced.
In order to aid the movement of the liquid, a
stirrer (preferably a magnetic stirrer) can additionally
2032~37
- 14 -
be provided.
The treatment method described above may also be
automated by having a suitable control device (e.g. a
PC) to regulate the in-flow and out-flow of liquids.
The apparatus may also be constructed so that a
plurality of containers as described above are provided,
in which several treatments may be carried out
separately from one another. Depending on the switching
of the PC, these treatments may run parallel or totally
independently. In numerous treatment methods for
electrophoresis carriers, particularly the staining
process, it is useful to provide storage containers for
relatively concentrated xeagent solutions in the
apparatus. Before being introduced into the treatment
container, these reagents are mixed directly in the
inlet pipe or in a special mixing chamber in the inlet
pipe with the corresponding quantity of solvent.
If a comparison is made between the conventional
manual treatment or the known apparatus, and the process
and apparatus according to the invention, the following
advantages become clear:
1. Compared with the conventional treatment by
carrying out the steps by hand, there is an
enormous time saving for the analyst.
In the procedure according to the invention, the
analyst has only to initiate the individual steps
by switching the regulating means over, but in
other respects the apparatus and treatment can be
left to run. This regulating process may also be
done by means of a computer. In the conventional
manual process, frequent interventions are
required. (The labour and time expended by the
analyst are particularly great in the longer
treatment steps, e.g. the rinsing operations
between the staining and decolorizing steps, in
2~32~8~
- 15 -
which at least 5 rinses are needed, each taking 5-
10 minutes.) In the process according to the
invention, by contrast, rinsing is carried out
continuously for 30 minutes without the need for
the analyst to intervene.
2. The time taken for the individual treatment steps
is also reduced, since the liquid in contact with
the surface of the electrophoresis carrier is
constantly renewed according to the invention by
the flow action.
3. In the process according to the invention,
initially a larger quantity of reagent is used,
owing to the continuous method of operation. The
consumption of reagent can be optimized by matching
the size of the apparatus to the size of the gel
which is to be treated. However, by regeneration
of the used reagent (recycling) the consu~ption of
reagent can be reduced to a level corresponding to
that of conventional methods.
4. The apparatus according to the invention is simple
in terms of construction and requires no
mechanically movable parts. The apparatus is
suitable for all electrophoresis carriers in common
use. Even electrophoresis carriers of different
sizes can be treated therein. It is also possible
to treat several electrophoresis carriers at the
same time, if they are secured in position (e.g. in
a frame) as mentioned above. The electrophoresis
carriers are simply placed in the container (or in
the frame by means of which they are then placed in
the container). There is no need for any laborious
mounting of the electrophoresis carriers, in for
example movable retaining means.
