Apparatus for electroelution

• US 5,340,449 A
• Filed: 10/23/1992
• Issued: 08/23/1994
• Est. Priority Date: 12/07/1990
• Status: Expired due to Term

• Current Assignee: Harvard Apparatus, Inc. (Harvard Bioscience, Inc.)
• Original Assignee: Ashok K. Shukla
• Inventors: Shukla, Ashok K.
• Primary Examiner(s): Niebling, John
• Assistant Examiner(s): Phasge, Arun S.
• Application Number:
  US07/965,862
• Time in Patent Office: 669 Days
• Field of Search: 204/180.1, 204/182.1, 204/182.3, 204/182.8, 204/299 R, 204/301
• US Class Current: 204/464
• CPC Class Codes: B01D 57/02 by electrophoresis treatment of water, waste water, sewage or sludge by electrophoresis C02F1/469; electrophoretic production of compounds or non-metals C25B7/00; investigating or analysing materials by using electrophoresis G01N27/26
• CPC Class Codes: B01D 61/28 Apparatus therefor
• CPC Class Codes: B01D 61/422 Electrodialysis
• CPC Class Codes: B01D 61/425 Electro-ultrafiltration
• CPC Class Codes: B01F 33/452 using independent floating stirring elements
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: G01N 2001/4016 being a selective membrane, e.g. dialysis or osmosis
• CPC Class Codes: G01N 27/4473 by electric means

Abstract

This invention involves the elution of macromolecules such as proteins, deoxyribonucleic acid(DNA), ribonucleic acid(RNA) from solid phase matrix such as polyacrylamide, agarose and membranes such as polyvinyl difluoride (PVDF) in an electric field. The elution chamber shown in FIG. 1 consists of two separate screws 9, 9a and attachable chamber 1. The screw well 8 holds the gel matrix containing the macromolecules and is separated by the bottom chamber 1 with a membrane 5 with a nominal molecular weight cutoff higher than the macromolecule. On the other hand, the macromolecule is retained in the well 1 by introducing another membrane 6 having a molecular weight cutoff lower than the molecular weight of the macromolecule. Alternatively, macromolecules separated on membrane, e.g. PVDF, are held in a Teflon or plastic membrane holder 11. This holder can fit inside well 8 in screw 9. In addition, chamber 1 contains a well 7. The macromolecules migrate through the electrophoretic field and are concentrated in well 7. Since the volume of the well 7 is predetermined (10 ul--one ml or more), results in concentration of the macromolecules. Next, the chamber 1 is unscrewed from the top screw 8 and closed with a screw without a hole 13, and the entire device is submerged into a dialysis buffer. Following dialysis for a given time, the fluid above the bottom screw 9a itself and the membrane beneath it are removed. The sample is taken out from hole 7. If further concentration of the sample is required, then following removal of the fluid above the screw 9a, the entire device is transferred to a vacuum desiccator and concentrated to any desired volume.

Claims

1. An electroelution device for the electroelution of macromolecules from a support, comprising a chamber and upper and lower releasably securable membrane retaining members, said chamber, and at least one of said upper and lower membrane retaining members having a through-hole for passage of a fluid, said chamber through-hole terminating at a membrane well at each of its ends and having a diameter less than that of said membrane wells, each of said membrane wells having a sidewall that is provided with a shoulder for receiving a membrane of a prescribed molecular weight porosity and a corresponding one of said membrane retaining members along a common axis such that the membrane is interposed between said shoulder and said membrane retaining member, said sidewalls and said membrane retaining members each having complementary-configured engagement surfaces for effecting engagement and retention of the respective membrane retaining members received therein.

2. The device of claim 1 wherein said membranes have molecular weight exclusions such that said macromolecules are retained within the through-hole of said chamber after electroelution.

3. The device of claim 2 wherein said membranes have different molecular weight exclusions.

4. The device of claim 1 which further comprises a holder for holding said support.

5. An electrophoresis tank containing the electroelution device of claim 1.

6. The electroelution device of claim 1 wherein the through-hole of at least one of said upper and lower membrane retaining members is tapered.

7. An electroelution device for the electroelution and separation of macromolecules from a support, the device comprising at least two chambers, a releasably securable upper membrane retaining member, a releasably securable lower membrane retaining member, and at least one connecting membrane retaining member, at least one of said upper and lower membrane retaining members, each of said chambers, and said connecting membrane retaining member each having a through-hole for passage of a fluid, the chamber through-holes having a diameter that exceeds that of the membrane retaining member through-holes, said chambers defining a membrane well at each end of said chamber through-hole having a sidewall that is provided with a shoulder for receiving a membrane of a prescribed molecular weight porosity and a corresponding one of said membrane retaining members along a common axis such that the membrane is interposed between said shoulder and said membrane retaining member, said sidewalls and said upper and lower membrane retaining members each having complementary-configured engagement surfaces for effecting engagement and retention of the respective membrane retaining members received within their corresponding membrane wells.

8. The device of claim 7 wherein said membranes have molecular weight exclusions such that said macromolecules are retained within the through-holes of said chambers after electroelution.

9. The device of claim 8 wherein said membranes have different molecular weight exclusions.

10. The device of claim 7 which further comprises a holder for holding said support.

11. An electrophoresis tank containing the electroelution device of claim 7.

12. The electroelution device of claim 7 wherein the through-hole of at least one of said upper and lower membrane retaining members is tapered.

13. A process for the electroelution of macromolecules from a support, comprising:

providing an electroelution device having a chamber and upper and lower membrane retaining members releasably securable within membrane wells formed int he chamber, the chamber and at least one of the membrane retaining members defining a through-hole for passage of a fluid, the membrane wells each including a sidewall having a configuration complementary to that of the membrane retaining members to engage and retain the membrane retaining members upon insertion into the wells and a shoulder for receiving an electroelution membrane of a prescribed molecular weight porosity and a corresponding one of the membrane retaining members along a common axis such that the membrane is interposed between the well shoulder and the retaining member;

inserting an electroelution membrane into at least one of said membrane wells;

inserting the electroelution device into an electrophoresis tank having a plate, a cathode and an anode;

inserting a support containing macromolecules to be separated into the through-hole of one of said membrane retaining members; and

applying an electric current across said electrophoresis tank to effect electroelution of said macromolecules from said support into the through-hole of said chamber.

14. The process according to claim 13, wherein the electroelution device comprises at least two chambers having membrane wells that are interconnectable by a connecting membrane retaining member, each of said chambers and said connecting membrane retaining member being provided with a through-hole for the passage of a fluid.

15. An electroelution device for immersion in an electrophoresis tank for the electroelution of macromolecules, said device comprising one or more chambers releasably sealable by an upper screw cap and a lower screw cap, each of said chambers and one (but only one) of said screw caps each having a through-hole for the passage of fluid.

16. A device as in claim 15, further comprising a magnet encased within said device for interaction with an external magnet to impart spinning motion to said device.

17. A device as in claim 15 comprising at least two chambers.