GB2147609A - Electrophoretic transfer - Google Patents

Abstract

Electrophoretic transfer apparatus has a support structure 32 pivotable about a horizontal axis in a buffer chamber. To transfer, for example, proteins or nucleic acids from a gel (where they have been separated by electrophoresis to a nitro-cellulose sheet, a suitable stack is formed on the horizontal support, under the surface of the buffer liquid. The support is then pivoted to the vertical position for electrophoretic transfer. Since the stack is formed under buffer, the problem of air entrapment between the layers is much reduced. The application also relates to electrophoretic transfer apparatus comprising a chamber for containing buffer liquid, a pair of electrodes 14, support means 24 for supporting sheet-like media for electrophoretic transfer, and guide means 12 extending transversely of the chamber, at least one of the electrodes being in sliding engagement with the guide means to vary the inter-electrode spacing. <IMAGE>

Description

SPECIFICATION Electrophoretic transfer apparatus This invention relates to electrophoresis and more particularly to apparatus for use in electrophoretic transfer.

This technique finds use in the transfer of proteins or nucleic acids from a gel - where they have previously been separated by electrophoresis - to a nitrocellulose sheet or the like which may then be divided to enable separate study of the individual components.

Using currently available apparatus, electrophoretic transfer is set up by assembling a stack from the gel, nitrocellulose and accompanying support structures and then positioning this stack in a chamber containing buffer liquid and electrodes. Problems are found to occur with air bubbles trapped in the stack, distorting or inhibiting the transfer process.

It one object of this invention to provide improved apparatus for use in electrophoretic transfer which substantially overcomes these problems.

Accordingly, the present invention consists, in one aspect, in apparatus for use in electrophoretic transfer, comprising a chamber for containing buffer liquid; electrode means disposed in the chamber and support means adapted to support first and second sheet-like media in the chamber for electrophoretic transfer from the one to the other, where the support means is movable in the chamber between a first orientation in which it is disposed for receipt of said media and a second orientation in which the supported media are suitably positioned for electrophoretic transfer.

Advantageously, the media are horizontal in said first orientation of the support means and vertical in the second orientation.

Preferably, the support means is pivotally mounted in the chamber for movement about a horizontal axis.

By the use of apparatus according to the invention, the gel and nitrocellulose, or other sheet-like media, can be assembled on the support means, under the buffer liquid. Once assembly is complete, the support means can, according to the preferred form of the invention, be pivoted to a vertical position for electrophoretic transfer to take place. Since assembly takes place underneath the buffer liquid, the problem of air bubbles is very much reduced if not eliminated. For operating convenience, the horizontal orientation of the support means can be arranged to be in a shallow depth of buffer liquid.

In apparatus which has previously been used for electrophoretic transfer, the electrodes are usually an integral part of the overall structure. The inter-electrode spacing is thus fixed and normally suitable for one supply voltage only. On the other hand, changes in the electric field established in the chamber can only be achieved by varying the supply voltage. Difficulties are encountered, moreover, in cleaning the chamber and the electrodes themselves. A futher drawback is that the type of electrode which is built into the apparatus may not be the ideal electrode for the particular transfer process to be undertaken and the chosen buffer liquid.

It is a further object of this invention to provide improved apparatus for use in electrophoretic transfer which alleviates some or all of the above problems.

Accordingly, the present invention consists, in a further aspect, in apparatus for use in electrophoretic transfer comprising a chamber for containing buffer liquid; a pair of electrodes disposed in the chamber; support means adapted to support first and second sheet-like media in the chamber for electrophoretic transfer from the one to the other and elongate electrode guide means extending transversely of the chamber, at least one of the electrodes being in sliding engagement with said guide means for movement relative to the chamber to vary the inter-electrode spacing.

Advantageously, the electrode guide means comprises a pair of guide rails, each electrode having a pair of support elements overlying the respective guide rails.

This invention will now be described by way of example with reference to the accompanying drawings in which: Figure 1 is a side view in section of apparatus according to this invention, Figure 2 is an end view in section of the apparatus of Fig. 1, and Figure 3 is a plan view in section of the apparatus in Fig. 1.

The apparatus shown in the drawings comprises a rectangular tank 10 formed of perspex or any other convenient material. The tank is provided with a suitably shaped lid which is not shown in the drawings. Along opposite walls of the tank 10, adjacent the upper edges thereof, are provided integral guide rails 12. A pair of electrode frames 14 are positioned in the chamber - one at each end - and are supported through the engagement of a pair of support lugs 1 6 with the respective guide rails 1 2. The electrode frame (seen best in Fig. 3) carries a serpentine platinum wire 1 8 connected electrically with a flexible supply cable 20 extending from the top of the electrode frame to one of a pair of supply terminals mounted in the lid (not shown).The electrode frames may be provided at their bottom edges with counterweights 22.

The pair of tank walls which are provided, as described above, with guide rails, are further provided each with a support block 24 projecting inwardly of the tank. Each support block extends parallel to the base of the tank from one end wall to a position just short of the opposing end wall. The blocks are positioned at approximately half the height of the tank and are formed, at their respective midpoints, with contoured recesses 26. Each recess has, as seen best in Fig. 1, a rectangular portion 28 and a semicircular portion 30. The function of this recess will become apparent as the manner of operation of the apparatus is later described.

The support structure for the gel and nitrocellulose sheet - shown generally at 32 in Fig. 1 - comprises a base plate 34 and a top plate 36 holding between them a stack formed of a SCOTCHBRITE mat: filter paper: gel: nitrocellulose sheet: filter paper: SCOTCHBRITE mat. The base and top plates are formed from perspex and have a series of closely spaced perforations. The base plate 34 has a pair of trunions 38 which, in the position shown in Fig. 1, engage with the rectangular portions of corresponding recesses 26. Along one edge of the plate there is provided a weighted member 40 the length of which exceeds that of the plate to define projecting lugs 42. In the position shown in Fig. 1, these projecting lugs rest on the free ends of respective support blocks 24.Near the side of the base plate opposite this weighted member, additional lugs 44 extend parallel with projecting lugs 42 and likewise engage the respective support blocks.

The top plate 36 is provided with a further weighted member 46 which in use overlies the member 40 of the base plate. Both the base plate 34 and top plate 36 are formed with small V-shaped notches which serve, in the assembled support structure 32 to locate straps, not shown, which hold the two plates together.

As shown in Fig. 3, the tank is provided with a buffer inlet 50 which is located towards the base of the tank and which communicates with a two way pump 52. This pump further communicates with a reservoir connection 54 and - through a valve 56 - with the base of an ion exchanger or activated charcoal filtration unit 58. The top of this filtration unit is connected with a buffer outlet 60 near the top of the tank.

The manner of operation of the described apparatus can now be understood.

Buffer liquid, which may be for example a methanol/water mixture, is pumped into the tank chamber from a suitable reservoir using the pump 52. The stack shown in Fig. 1 is then assembled by positioning the layers individually on the base plate. The stack is completed by the top plate which is then secured to the base plate by the use of encircling straps. It will be appreciated that as the stack is assembled underneath the surface of the buffer liquid, the problems of entrapped air bubbles are very much reduced if not eliminated.

With the assembly of the stack complete, the base plate is displaced slightly to the left (as shown in Fig. 1) so moving the trunions 38 into the semicircular portions of the recesses 26. This same movement brings the projecting lugs 40 free of the support blocks 24 enabling the stack to pivot anticlockwise (as shown in Fig. 1) about the axis defined by the trunions and under the action of the weighted members 40, 46. In the vertical position shown in dotted outline in Fig. 1, the gel and nitrocellulose are correctly disposed relative to the electrodes 14, for electrophoretic transfer.

To reduce the problem of contamination of the buffer with, for example, SDS remaining after electrophoretic separation in the gel, the buffer may be recirculated through the filtration unit, either intermittently or continuously.

If desired, the filtration unit can be combined with a cooling unit to prevent an unacceptable temperature rise in the buffer.

To accommodate different supply voltages, or to vary the applied field, either or both of the electrode frames may be moved transversely of the tank with the support lugs 16 sliding along the guide rails 12. When it is necessary to clean the tank, the electrode frames may be simply lifted out. This facility has the further advantage of enabling ready interchange between different electrode types.

It will be appreciated that this invention has been described by way of example only and a variety of modifications are possible without departing from the scope of the invention. For example, the described arrangement could be replaced by other means for supporting the gel and nitrocellulose or other transfer media.

Similarly, the described arrangement of trunions and cooperating recesses could be replaced by other pivotal mountings or indeed by other means of bringing the support from the horizontal assembly position to the vertical transfer position. Projections on the support plates could for example cooperate with contoured tracks formed in opposing tank walls.

A horizontal assembly position is clearly desirable for ease of positioning the individual layers in the stack whilst a vertical transfer position has the advantage that gases evolved during the transfer process are free to rise. In certain situations, nonetheless, alternative orientations may be selected.

Claims (1)

1. Apparatus for use in electrophoretic transfer, comprising a chamber for containing buffer liquid; electrode means disposed in the chamber and support means adapted to support first and second sheet-like media in the chamber for electrophoretic transfer from the one to the other, wherein the support means is movable in the chamber between a first orientation in which it is disposed for receipt of said media and a second orientation in