Methods for separation and immuno-detection of biomolecules, and apparatus related thereto

Abstract

Disclosed are methods and apparatus for separation of biomolecules via two-dimensional gel electrophoresis, methods and apparatus for immunoblotting separated biomolecules, and methods for the use of biomolecules processed via the methods and apparatus of the present invention, including use in a clinical setting. The methods and apparatus for separation of biomolecules via two-dimensional gel comprises vertical agarose gel electrophoresis in the first dimension, and the electrophoresis of a novel non-denaturing 3-35% concave gradient polyacrylamide gel in the second dimension. This novel gel can be cast in a modified gel caster that can facilitate the pouring of multiple gels simultaneously. The methods and apparatus for immunoblotting are useful with any type of immunoblotting, including Western blot, Northern blot, and Southern blot analyses. These methods and apparatus provide safe, efficient and cost-effective immunoblots, while facilitating the reduction of exposure to toxic or radioactive materials, as well as the disposal of those materials.

We claim: 
     
         1 . A closed-chamber apparatus for immunoblotting, comprising:
 a. an incubation chamber, comprising a body having interior and exterior surfaces and two open opposing ends, and comprising a plurality of membranes, said incubation chamber capable of containing incubation media,   b. a rotating means, and   c. a driving means in communication with said rotating means;   said rotating means providing rotation perpendicular to the plane of said plurality of membranes.   
     
     
         2 . A closed-chamber apparatus for immunoblotting according to  claim 1 , said closed-chamber apparatus comprising a peristaltic pump, and said rotating means being a roller assembly comprising at least two rolls capable of rotation. 
     
     
         3 . A closed-chamber apparatus for immunoblotting according to  claim 2 , said incubation chamber comprising:
 a. two covers, each cover configured to engage each of two open opposing ends of said incubation chamber body,   b. two O'rings,   c. two shut-off valves,   d. optionally, at least one fluid transfer means, said at least one fluid transfer means capable of engaging with at least one of said two shut-off valves;   said plurality of membranes comprising upto 5 membranes.   
     
     
         4 . A closed-chamber apparatus for immunoblotting according to  claim 3 , said incubation chamber comprising:
 a. a membrane holding means,   b. a fastening means, and   c. at least one rod;   each of said two covers comprising each of said two shut-off valves,   said incubation chamber being in rotatable communication with the at least two rolls of said roller assembly.   
     
     
         5 . A closed-chamber apparatus for immunoblotting according to  claim 2 , said incubation chamber body comprising a cylindrical shape. 
     
     
         6 . A closed-chamber apparatus for immunoblotting according to  claim 5 , comprising a plurality of additional incubation chambers, each of the plurality of additional incubation chambers being in rotatable communication with the at least two rolls of said roller assembly. 
     
     
         7 . A closed-chamber apparatus for immunoblotting according to  claim 2 , comprising:
 a. an outer component comprising a body having interior and exterior surfaces and two open opposing ends, said outer component configured to house said incubation chamber,   b. two covers, each cover configured to engage each of two open opposing ends of said outer component body,   c. two shut-off valves,   d. optionally, at least one fluid transfer means, said at least one fluid transfer means capable of engaging with at least one of said two shut-off valves;   said incubation chamber being located inside of said outer component and at least a portion of the exterior surface of said incubation chamber body being in communication with at least a portion of the interior surface of said outer component, said outer component being in rotatable communication with the at least two rolls of said roller assembly.   
     
     
         8 . A closed-chamber apparatus for immunoblotting according to  claim 7 , comprising a membrane holder configured to hold a plurality of membranes, said plurality of membranes comprising upto 100 membranes, said membrane holder comprising:
 a. at least one rod,   b. a quantity of separation means to accommodate the upto 100 membranes, at least one of said separation means being disposed near the perimeter of each membrane,   c. at least one horizontal mixing means.   
     
     
         9 . A closed-chamber apparatus for immunoblotting according to  claim 7 , comprising:
 a. an O'ring, being disposed between one of said two covers and one of the two open opposing ends of said outer component body, and forming a closure combination, and   b. at least one clamping means, said at least one clamping means being in clamping communication with said closure combination and forming a hermetic seal at one end of said outer component body.   
     
     
         10 . A closed-chamber apparatus for immunoblotting according to  claim 7 , said outer component comprising a cylindrical shape. 
     
     
         11 . A closed-chamber apparatus for immunoblotting according to  claim 7 , said incubation chamber body comprising a square shape in the x- and y-axis. 
     
     
         12 . A two-dimensional gel electrophoresis method, comprising vertical agarose gel electrophoresis in the first dimension, said method being utilized for the separation of biomolecules. 
     
     
         13 . A two-dimensional gel electrophoresis method according to  claim 12 , comprising non-denaturing 3-35% concave gradient polyacrylamide gel electrophoresis in the second dimension. 
     
     
         14 . A two-dimensional gel electrophoresis method according to  claim 13 , said biomolecules being proteins or lipoproteins. 
     
     
         15 . A method for immunoblotting separated biomolecules, comprising:
 a. incubation of separated biomolecules in communication with a stack of membranes in a closed chamber, said closed chamber comprising incubation media comprising ligands monospecific to the separated biomolecules of interest, and   b. mixing of the incubation media, said mixing comprising rotation of said closed chamber, said rotation being perpendicular to the plane of said stack of membranes;   said stack of membranes comprising up to 100 membranes, and   said closed chamber being hermetically sealed.   
     
     
         16 . A method for immunoblotting separated biomolecules according to  claim 15 , said closed chamber having a cylindrical shape. 
     
     
         17 . A method for immunoblotting separated biomolecules according to  claim 15 , comprising placing said closed chamber inside of an outer component configured to receive said closed chamber. 
     
     
         18 . A method for immunoblotting separated biomolecules according to  claim 17 , said closed chamber having a square shape in the x- and y-axis, and said outer component having a cylindrical shape. 
     
     
         19 . A method for immunoblotting separated biomolecules according to  claim 17 , said closed chamber comprising a mixing means, said mixing comprising horizontal mixing via said mixing means, and said membranes in the stack being separated from each other via at least one separation means. 
     
     
         20 . A method for immunoblotting separated biomolecules according to  claim 15 , said method comprising identification of the separated biomolecules. 
     
     
         21 . A method for immunoblotting separated biomolecules according to  claim 20 , said separated biomolecules being biomolecules separated via two-dimensional gel electrophoresis. 
     
     
         22 . A method for immunoblotting separated biomolecules according to  claim 21 , said two-dimensional gel electrophoresis comprising vertical agarose gel electrophoresis in the first dimension, and non-denaturing 3-35% concave gradient polyacrylamide gel electrophoresis in the second dimension. 
     
     
         23 . A method for immunoblotting separated biomolecules according to  claim 22 , said separated biomolecules being HDL particles. 
     
     
         24 . A method of therapy, comprising the use of separated biomolecules identified according to the method of  claim 22 , said use being diagnosis of a patient's condition, treatment of a patient's condition, or a combination thereof. 
     
     
         25 . A multiple gel caster apparatus to facilitate simultaneous pouring of gels, comprising:
 a. a frame configured to accept a plurality of gel cassettes,   b. a lid configured for securely closing said frame, said lid being in communication with said frame,   c. a port in communication with said lid,   d. a plurality of gel cassettes disposed in said frame, and   e. a heat dissipating means disposed between said plurality of gel cassettes.