US3534747A - Destainer for disc electrophoresis rods utilizing circulating liquid - Google Patents

Description

United States Patent 6m lll l mu 3 2,319,531 5/1943 Brownetal...........i....... 2,616,095 11/1952 Stuckey......

Peter Stanton Hoefer San Francisco, California [72] Inventor 134/171X 134/111X 134/147X Primary Examiner-Robert L. Bleutge Atr0mey-Flehr, Hohbach, Test, Albritton and Herbert Scientific Instruments, San Francisco, California .mvw .l m e" a m h 6W0 ueo BLK 556 556 999 111 802 6 2 071 503 29 223 AFPA [54] DESTAINER FOR DISC ELECTROPHORESIS RODS UTILIZING CIRCULATING LIQUID 15 Claims, 4 Drawing Figs.

ABSTRACT: A destainer for disc electrophoresis rods which are contained in tubes including a cylindrical container for holding acetic acid which has a magnetic stirring rod at its base to set up a vortex circulation. The rods are placed in a flange-type holder which mates with the walls of the container to force substantially all upward circulating liquid through the I t, tubes. The holder includes an inner column having openings at References C'ted its top to allow for downward circulation of the liquids UNITED STATES PATENTS through both an activated charcoal filter and for free move- 3/1938 Adams et a1. ment down toward the stirring rod.

Patented Oct. 20, 1970 3,534,747

INVENTOR.

PETER s. HOEFER i, M M M v m ATTORNEYS DESTAINER FOR DISC ELECTROPIIORESIS RODS UTILIZING CIRCULATING LIQUID The present invention is directed to a destainer and more specifically to a destainer for use with disc electrophoresis rods.

The destaining of disc acrylamide gels is essential to complete the analysis of proteins which have been separated by disc electrophoresis. Disc electrophoresis is a method for separating the ionic components of a mixture, usually proteins, into separate, distinguishable bands in an acrylamide gel of rodlike configuration. After the ionic components have been separated within the gel, they are not visible to the eye until they have been stained and fixed, usually with Amido Schwarz stain. Because the stain permeates the entire gel, rendering it a dense, opaque blue-black color, the surplus stain, that is, all stain which is not held by the ionic bands, must be removed before the bands themselves become visible. This operation can be done in two ways: one is by electrophoresis in which direct current is passed through the gel, using dilute acetic acid as an electrolyte; the other is by a diffusion or leaching method in which the gels are bathed in circulating dilute acetic acid. The diffusion method requires that the stain be absorbed as it is given up by the gels.

The diffusion process as now practiced requires a length of time measured in days to complete such absorption of the stain.

It is therefore a general object of the present invention to provide an improved destainer for electrophoresis rods.

It is another object of the invention to provide for such destaining in a relatively short period of time.

It is another object of the invention to provide a destainer which has improved liquid circulation.

In accordance with the above objects there is provided a destainer for disc electrophoresis rods contained in tubes which utilizes the circulating liquid. The destainer includes a liquid container with means within the container defining a first annular space. A plurality of rod receiving tubes are supported in this space. Means are provided for circulating liquid through the annular space and the tubes. At least a portion of the liquid flow is filtered.

In another aspect of the invention stirring means are provided for maintaining the liquid circulation. The stirring means include a bar of magnetic material pivoted on the bottom of the container and coupled magnetically to an externally applied magnetic field which causes the bar to rotate.

These and other objects of the invention will become more clearly apparent from the following description.

Referring to the figures:

FIG. 1 is a perspective view of a destainer embodying the present invention which is exploded and partially cut away;

FIG. 2 is a cross-sectional view of a destainer embodying the present invention which has been simplified to better explain the invention;

FIG. 3 is a top view of FIG. 2; and

FIG. 4 is an enlarged perspective view of one of the tubes of the present invention which contains an electrophoresis rod.

Referring now to FIG. 1, the destainer includes a liquid container 11 having a predetermined inner diameter 12. On the bottom 13 of the container are stirring means in the form of an ellipsoidal bar 14 including magnetic material which is located in an indentation 13a (FIG. 2) of bottom 13. Bar I i provides stirring action for liquid in container 11. It is rotated by an externally applied rotating magnetic field supplied from a source 16 (FIG. 2). The magnetic stirring source 16 as utilized in the present invention is readily available from any chemical supply company.

Referring now again to FIG. 1, an inner column 17 is provided which is coaxial with the cylindrical container 11 and has an outer diameter less than the inner diameter 12; of the container so it may fit therein and provide an annular space there between. Inner column I7 is divided into two separate portions; the bottom portion 17a is affixed to bottom I3 and has elliptical ports 18 to allow, for the passage of liquid from the inner portion of the column to the remainder of the container. Bottom column portion 17a also includes a top retaining ring cap l9 having a aperture 21. Aperture 2i and ports I8 are dimensioned so as to normally retain ellipsoid stirring bar 14 within this portion of the column; in other words, during inactive periods of the device while. for example, it is being moved from one location to another, the stirrer i4- is free to roll within the confines of the inner column without falling through any of the ports, and if the device is inverted, the aperature 21 is smaller than the long axis of the stirring bar to prevent it from falling out of its enclosure.

Positioned on top of the retaining ring 19 are three posts 22 (see also FIG. 2) which support a filter 23 whose conical end 24 rests upon the posts. Sufficient space is provided between the cone 24 and aperture 2i to allow for circulation of liquid between the two. Cone 24 is truncated with the tip of the cone being located in close juxtaposition to stirring rod 14. The remainder of the filter is a cylinder containing particulate filter material 2a which is retained by a disc of porous material 2'? placed in the top of filter 23. In the case of the present invention, the filter material is preferably activated charcoal.

A single tube used in the present destainer is shown in FIG. 4. The tube consists of a tubular glass portion 31 and a lower enclosure 32 with a sleeve 34) and a perforated bottom 33 to allow for circulation of liquid there through from the bottom and out of the top. Placed in glass portion 3i is an electrophoresis rod 34 which has recently been stained. Rod 34 is of a smaller diameter than tube 3i to allow for the free circulation of the destaining liquid.

Supporting means for tubes 31 include a flange 36 extending from the lower end of column portion 17b and having an outer diameter substantially equal to inner diameter E2 of container 11. The flange 36 includes apertures 37 for receiving the lower enclosure 32 of tubes 31. When in its proper location, the flange rests on retainer ring 19 of the lower portion 17a of inner column I7. Posts 35 extending from the bottom of annulus 3b support the holder during the initial placement of the tubes in it.

Top portion ll7b of inner column I7 includes horizontal elongated openings 38 near the upper end of the inner column. These openings allow for the flow of liquid from the annular space between column and container into the inner column.

Lastly, a perforated retainer ring 39 is rested on the top of tubes iii to retain rods 34 within the tubes during the upward circulation of the liquid. The entire container 11 is enclosed by a top Ml which rests upon inner column 17.

OPERATION Referring now especially to FIG. 2, after the tubes having electrophoresis rods are placed in the holder and the container filled, for example, with dilute acetic acid, the stirrer 14 is activated. This creates a vortex type of circulation where the liquid flows from the bottom of the container up along its periphery through tubes 31 through openings 30 and thence down again through the inner column 17. Because of the design of the device with a flange 36 of substantially the same diameter as the inner diameter of container II, the only substantial upward movement of liquid which may occur is through the tubes 31. as indicated by the arrows. Since the electrophoresis rods 34 are slightly smaller than the diameter of tubes 3i, this permits circulation of the liquid past the rods to wash away the uniixed stain.

In the return movement of the liquid downward as indicated by the arrows, the liquid flows through openings 38 with the great majority of the liquid flowing between the wall of the inner column l7 and the wall of filter 23 down through aperture 21 to the stirrer. Part of the downward flow, however, is constantly filtered through 23 which absorbs the stain. This filtered portion of the flow is maintained in part by a suction created by the action of stirring rod 14 located directly beneath the tip of cone 24 of the filter. By only filtering part of the liquid, the downward flow meets little resistance and,

therefore, sustains a high volume of circulation. In this manner destaining can be accomplished in several hours.

Thus, the destainer of the present invention provides a device which due to the inherent high volume of liquid circulation rapidly destains electrophoresis rods. Moreover, with a magnetic stirring bar the necessity of cumbersome external pumps are eliminated and maximum advantage is taken of the magnetic stirring by providing that substantially all of the upward flow of liquid occurs only through the tubes to achieve maximum destaining action.

lclaim:

l. A destainer for disc electrophoresis rods contained in tubes which utilizes a circulating liquid comprising a liquid container, means within said container defining a first annular space, annular flange means for supporting within such annular space a plurality of rods receiving tubes, filter means disposed in said container defining a second annular space through which said liquid flows, means for circulating liquid through said first and second annular spaces said flange means being dimensioned to occupy substantially all of a cross section of said first annular space said tubes extending through said flange means and having bottom perforations for causing substantially all of said circulating liquid to pass through said tubes.

2. A destainer as in claim 1 where said circulating means include a bar containing magnetic material pivoted on the bottom of said container and coupled magnetically to an externally applied magnetic field which causes it to rotate.

3. A destainer for disc electrophoresis rods contained in tubes which utilizes a circulating liquid comprising, a liquid container with a bottom and having a predetermined inner diameter, liquid circulating means centrally located on said bottom, an inner column coaxial with said container defining an annular space with said column and having lower ports in close proximity to said container bottom, said ports coupling the interior of said inner column to said annular space, a filter column within said inner column having one end in juxtaposition with said liquid circulating means, holding means for said tubes including a flange extending from said inner column, having an outer diameter substantially equal to said inner diameter of said container, said flange being located above said ports and having apertures formed therein for receiving said tubes, the interior of said tubes forming a path for said circulating liquid.

4. A destainer as in claim 3 where said tubes are maintained in said flange parallel to the axis of said inner column.

5. A destainer as in claim 3 where said tubes include a lower enclosure adapted to seat within the apertures in said flange said enclosures having perforations in them to allow the circulation of said liquid.

6. A destainer as in claim 3 including an annular retainer ring which is perforated to allow the circulation of liquid and which is rested on top of said tubes to maintain any material which is in said tubes during said liquid circulation.

7. A destainer as in claim 3 where said inner column includes openings in its top portion which are located above the tops of said tubes to allow for liquid flow.

8. A destainer as in claim 8 where said openings are elongated and horizontal.

9. A destainer as in claim 3 where said circulation means has a center of rotation and where said filter column terminates in a truncated cone with the tip of said cone located in close juxtaposition to said center of rotation.

10. A destainer as in claim 3 where said inner column includes a first portion and a second portion with said second portion including said ports and being affixed to the bottom of said container and said first portion being affixed to said flange, said first portion being removable from said container for easy placement of said tubes in said flange.

11. A destainer as in claim 10 where said circulating means includes a unitary bar which is free to move in any direction during nonactivated periods of said destainer and where said second ortion of said column includes an upper aperture which a ong with said ports are dimensioned to retain said bar within said second portion of said column.

12. A destainer as in claim 3 where said filter column is filled with particulate filter material, said column having a top opening with a porous retainer in said top opening.

13. A destainer as in claim 12 where said particulate materials is activated charcoal.

14. A destainer as in claim 3 where said circulation means includes an ellipsoidal stirring bar of magnetic material pivoted in an indentation in said container bottom and coupled magnetically to an externally applied magnetic field which causes said bar to rotate.

15. A destainer as in claim 3 where said circulating liquid is dilute acetic acid.

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