Automated system for two-dimensional electrophoresis

• US 6,245,206 B1
• Filed: 06/24/1999
• Issued: 06/12/2001
• 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): Nogueroila, Alex
• Application Number:
  US09/339,164
• Time in Patent Office: 719 Days
• Field of Search: 356/344, 204/456, 204/457, 204/461, 204/462, 204/466, 204/450, 204/600, 204/606, 204/607, 204/608, 204/612, 204/613, 204/616, 204/619, 204/620, 702/22, 436/46, 436/47, 436/48, 382/128, 425/135, 425/139
• US Class Current: 204/461
• 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. A method of scanning a stained clectrophoresis gel, said method comprises the steps of:

a) immersing said stained gel in a thin planar cavity filled with a liquid having a refractive index similar to that of said gel, wherein at least one side of said stained gel contacts said liquid;

b) introducing an illuminating light into said cavity approximately in the plane of said stained gel, wherein said illuminating light is substantially internally reflected in said cavity and thereby prevented from exiting said cavity normal to a plane of said stained gel; and

c) positioning an optical scanner such that said stained gel is viewed from outside of said cavity along a line of sight normal to said stained gel.

2. The method according to claim 1, wherein said cavity is a shallow horizontal depression filled with said liquid, wherein said liquid is aqueous.

3. The method according to claim 1 further comprising the step of positioning a light absorbing surface on an opposite side of said gel from said optical scanner, wherein said gel is stained with a fluorescent dye.

4. The method according to claim 3, wherein said illuminating light comprises a spectrum enriched in an appropriate excitation wavelength of said dye and depleted of an appropriate emission wavelength of said dye, and said light impinges on said optical scanner after passage through a filter which preferentially absorbs said excitation wavelength and transmits said appropriate emission wavelength of said dye.

5. The method according to claim 1 further comprising the steps of:

a) positioning a light absorbing surface on an opposite side of said gel from said scanner; and

b) differentiating a plurality of stained features on said gel from background by a greater or a lesser scattering of said illuminating light in a direction of said scanner, wherein said gel is stained with a particulate stain.

6. A method of establishing relative quantitation of proteins resolved in an clectrophoresis gel, said method comprising steps of:

a) staining said gel such that a plurality of optical properties of a plurality of protein-containing regions in said gel are progressively changed during a period of time;

b) optically scanning the stained gel from step (a) two or more times during said period wherein said scanning comprises i) immersing said stained gel in a thin planar cavity filled with a liquid having a refractive index similar to that of said stained gel, wherein at least one side of said gel contacts said liquid ii) introducing an illuminating light into said cavity approximately in the plane of said stained gel, wherein said illuminating light is substantially internally reflected in said cavity and thereby prevented from exiting said cavity normal to a plane of said stained gel, and iii) positioning, an optical scanner such that said stained gel is viewed from outside of said cavity along a line of sight normal to said stained gel;

c) measuring each of said optical properties as a function of time and recording a time sequence for said optical properties;

d) deriving a mathematical index from said time sequence; and

e) calculating relative protein abundance of said stained gel or other useful property of said stained gel from said index.

7. The method according to claim 6, wherein said gel is stained using i) a silver-based stain, ii) a negative stain based on interaction of A) a detergent or a plurality of ions in said gel with B) a plurality of copper ions or zinc ions in said gel, iii) a Schiff stain for carbohydrates, or iv) other kinetic stain, wherein a plurality of rates of diffusion of a plurality of reactants or a plurality of rates of chemical reactions determine said period of time.

8. The method according to claim 7, wherein said optical properties comprise transmittance, absorbance, fluorescence, luminescence, light scatter or refractive index as a function of time.

9. The method according to claim 7, wherein said mathematical index comprises a maximum rate of change of said optical properties as a function of time, or an increment of time relative to a fixed time at which a given change in said optical properties is detected, or a combination of said rate of change and said increment with one or more of said optical properties.

10. A method of establishing relative quantitation of proteins resolved in an electrophoresis gel wherein

a) staining said gel by two or more staining procedures to reveal said proteins;

b) optically scanning said the stained gel from step (a) to detect a plurality of stain results after or during each of said staining procedure, wherein said scanning comprises i) immersing said stained gel in a thin planar cavity filled with a liquid having a refractive index similar to that of said stained gel, wherein at least one side of said stained gel contacts said liquid, ii) introducing an illuminating light into said cavity approximately in the plane of said stained gel, wherein said illuminating light is substantially internally reflected in said cavity and thereby prevented from exiting said cavity normal to a plane of said gel, and iii) positioning an optical scanner such that said gel is viewed from outside of said cavity along a line of sight normal to said gel; and

c) collectively interpreting said stain results by means of a plurality of mathematical equations to determine a single measure of protein abundance.