Automated system for two-dimensional electrophoresis

• US 6,123,821 A
• Filed: 09/27/1999
• Issued: 09/26/2000
• Est. Priority Date: 06/24/1997
• Status: Expired due to Fees

• Current Assignee: Large Scale Biology Corporation
• Original Assignee: Large Scale Biology Corporation
• Inventors: Goodman, Jack, Anderson, N. Leigh, Anderson, Norman G.
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): NOGUEROLA, ALEXANDER STEPHAN
• Application Number:
  US09/405,213
• Time in Patent Office: 365 Days
• Field of Search: 204/450, 204/456, 204/457, 204/459, 204/465, 204/466, 204/600, 204/606, 204/607, 204/610, 204/619, 204/620, 264/259, 264/299, 264/308, 264/331.18, 425/122, 425/129.1, 425/117, 425/449, 425/436 R, 425/444, 435/287.3
• US Class Current: 204/456
• CPC Class Codes: G01N 2001/288 Filter punches
• CPC Class Codes: G01N 27/44773 Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
• CPC Class Codes: G01N 27/44782 of a plurality of samples
• CPC Class Codes: G01N 27/44795 Isoelectric focusing
• CPC Class Codes: Y10T 436/113332 with conveyance of sample along a test line in a container or rack
• CPC Class Codes: Y10T 436/25 including sample preparation
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

The present invention provides an integrated, fully automated, high-throughput system for two-dimensional electrophoresis comprised of gel-making machines, gel processing machines, gel compositions and geometries, gel handling systems, sample preparation systems, software and methods. The system is capable of continuous operation at high-throughput to allow construction of large quantitative data sets.

Claims

1. An apparatus for preparing a gel medium for separating a plurality of protein molecules, said apparatus comprising:

a) a movable mold half which, when placed near a gel-binding material, forms a mold cavity;

b) means for moving said movable mold half;

c) means for transporting said gel-binding material; and

d) means for dispensing a polymerizable gel mixture into said mold cavity.

2. The apparatus of claim 1 wherein an O-ring is between said movable mold half and said gel-binding material and wherein said O-ring contacts both said movable mold half and said gel-binding material.

3. The apparatus of claim 1 therein said means for dispensing comprises a means for dispensing a variable composition of said polymerizable gel mixture, wherein a gradient of said gel mixture is formed.

4. The apparatus of claim 1 wherein said gel mixture chemically bonds to said gel-binding material.

5. The apparatus according to claim 1, wherein said apparatus further comprises means for removing said gel-binding material from said mold cavity.

6. The apparatus according to claim 1 further comprising means for controlling temperature of said gel mixture within said mold cavity.

7. A method of preparing a gel medium for separating a plurality of protein molecules, wherein said method comprises steps of:

a) attaching a gel-binding material to a longitudinal mold cavity;

b) dispensing a variable composition of a polymerizable gel mixture into said mold cavity through a delivery tube having an open end, wherein said open end is inserted into said mold cavity, wherein said gel mixture comprises a gradient and wherein said gel mixture becomes chemically bonded to said backing material;

c) withdrawing said delivery tube from said mold cavity during said dispensing whereby said open end of said delivery tube remains at or just above the rising meniscus of said gel mixture in said mold cavity; and

d) removing said gel-binding material and said gel mixture from said mold cavity, wherein steps (b) and (c) are computer controlled.

8. The method according to claim 7, wherein said gel medium is prepared and used for the analysis of a single sample, wherein said sample comprises of said protein molecules.

9. The method according to claim 7, wherein said gel medium has a pH gradient along its length, rendering said gel medium suitable for use as an IPG gel.

10. The method according to claim 7, wherein said delivery tube is washed with a non-polymerizable solvent composition thereby preventing polymerization of said gel mixture in said delivery tube.

11. An apparatus for processing a gel medium for separating a plurality of protein molecules wherein said gel medium is fixed to a backing material, wherein said apparatus comprises:

a) a plurality of stations;

b) means for loading said gel medium with said protein molecules;

c) means for reversibly grasping said backing material; and

d) means for transporting said gel medium to said stations,

wherein one or more of said stations comprise i) means for application of a voltage longitudinally across said gel medium and ii) a feature selected from

     1) means for washing, dehydrating and rehydrating said gel medium;

     2) means for holding said gel medium in place at each of said stations, or

     3) a plurality of slots into which a plurality of gel mediums may be inserted.

12. A gel medium for separating a plurality of protein molecules, wherein said gel medium comprises two regions of distinct geometry wherein a first region is essentially planar and provides a medium for separating a plurality of macromolecules and a second region is non-planar and provides a means for suspending said gel by an edge in liquid or gaseous surroundings.

13. The gel medium according to claim 12, wherein said second region is formed with projections or indentations which facilitate grasping of said second region when transporting or holding said gel medium.

14. A programmable system for separating a plurality of protein molecules in a plurality of gel mediums respectively, said system comprising:

a) means for preparing said gel mediums;

b) means for loading each of said gel mediums with said protein molecules;

c) means for separating said protein molecules in each of said gel mediums;

d) means for staining or otherwise revealing proteins in each of said gels; and

e) means for scanning each of said gel mediums to detect resolved macromolecules.

15. The programmable system according to claim 14 further comprising a plurality of stations.

16. The programmable system according to claim 15, wherein each of said gel mediums is assigned thereto a plurality of time intervals for each of said stations respectively.

17. The programmable system according to claim 16 further comprising means for scheduling said time intervals for each of said stations respectively.

18. The programmable system according to claim 17, wherein said means for scheduling is asynchronous.

19. The programmable system according to claim 16 wherein said time intervals assigned to at least one of said gel mediums is different from said time intervals assigned to at least a second of said gel mediums.

20. The programmable system according to claim 16 further comprising a database, wherein a first plurality of steps and a first plurality of execution times based on said time intervals for a first sample are entered into said database, and a second plurality of steps and a second plurality of execution times based on said time intervals for a second sample are entered into said database, said second plurality of steps including a start time delay calculated so as to prevent any action required for said second sample from interfering with any action required for said first sample, and said system retrieves said first and second plurality of steps and said first and second plurality of execution times from said databases and carries out said first and second plurality of steps in time order.

21. The programmable system according to claim 15, wherein each of said gel mediums is assigned thereto a plurality of parameters for separating said protein molecules in each of said gel mediums.

22. The programmable system according to claim 21 further comprising means for controlling said plurality of parameters assigned to each of said gel mediums.

23. The programmable system according to claim 21 wherein said parameters comprise volt-hours.

24. The programmable system according to claim 21 wherein said parameters comprise a pH gradient of said gel mediums.

25. The programmable system according to claim 21, wherein said parameters assigned to at least one of said gel mediums are different from said parameters assigned to at least a second of said gel mediums.