Solid phase isolation of proteins, nucleic acids and other macromolecules
View Patent ↗The invention is a method for the isolation of molecules of interest using tubes in which at least a portion of the inner walls of the tube are coated with microbeads that are coated with a capture reagent to bind the molecule of interest. The microbeads may be glass or polymer beads. The invention is a method and apparatus for preparation of the tubes for use in the method of the invention. The invention is a method for determining ratios of guanine nucleotides bound to guanine-nucleotide binding proteins.
1. A method for isolating a macromolecule, comprising:
partially melting an inner wall of a test tube;
contacting the partially melted inner wall of the test tube with a plurality of beads after partially melting the inner wall of the test tube;
coating the beads with a capture reagent of the macromolecule; and
incubating the coated beads with a solution containing the macromolecule under conditions to allow binding of the macromolecule to the capture reagent, thereby isolating the macromolecule.
2. The method of claim 1 , wherein the beads are glass microbeads.
3. The method of claim 1 , where in the beads are polymer microbeads.
4. The method of claim 3 , wherein the microbeads are agarose.
5. The method of claim 1 , wherein the capture reagent is attached to the beads by at least one linker molecule.
6. The method of claim 5 , wherein the linker molecule is aminopropyltriethyoxysilane.
7. The method of claim 5 , wherein the linker molecule is cyanogen bromide.
8. The method of claim 5 , wherein in the linker molecule is a chemical cross-linking agent.
9. The method of claim 8 , wherein the cross-linking agent is dimethyl suberimidate.
10. The method of claim 5 , wherein the linker molecule is an antibody.
11. The method of claim 5 , wherein the linker molecule is protein A or protein G.
12. The method of claim 1 , further comprising washing the coated beads with the bound macromolecule with a wash buffer to remove unbound material while maintaining binding of the macromolecule to the capture reagent; and eluting the macromolecule from the capture reagent.
13. A method for isolating guanine nucleotide-binding proteins for determination of guanine nucleotide ratios comprising:
partially melting an inner wall of a test tube;
contacting the partially melted inner wall of the test tube with a plurality of glass beads wherein the beads have a surface after partially melting the inner wall of the test tube;
reacting the beads with an agent to modify the surface of the beads to provide a plurality of free amino groups;
reacting the free amino groups on the beads with a bifunctional amine cross-linker to provide a plurality of sites for binding a guanine nucleotide-binding protein binding partner; and
incubating the coated beads with a solution containing the guanine nucleotide-binding protein under conditions to allow binding of the guanine nucleotide-binding protein to the binding partner while inhibiting nucleotide hydrolysis or release, thereby isolating the guanine nucleotide-binding protein.
14. A method comprising:
heating a plurality of beads to a temperature sufficient to partially melt an inner wall of a tube;
contacting the heated beads with the inner wall of the tube after heating the plurality of beads to the temperature sufficient to partially melt the inner wall of the tube;
coating the beads with a capture reagent of a macromolecule; and
incubating the coated beads with a solution containing the macromolecule under conditions to allow binding of the macromolecule to the capture reagent.
15. The method of claim 1 , wherein the inner wall of the tube is partially melted using a heat gun.
16. The method of claim 1 , wherein the inner wall of the tube is partially melted using infrared irradiation.
17. The method of claim 1 , wherein the inner wall of the tube is partially melted using a filament.
18. The method of claim 1 , wherein the tube is a microcentrifuge tube.
19. The method of claim 1 , wherein the tube comprises a polymeric material, polypropylene, or polystyrene.
20. The method of claim 1 , wherein the macromolecule is a protein, peptide, nucleic acid, carbohydrate, or polymer.
21. The method of claim 1 , wherein the macromolecule is a polynucleotide.
22. The method as in claim 12 , wherein the wash buffer is removed by inversion of the tube.
23. The method of claim 13 , further comprising washing the coated beads with the bound guanine nucleotide-binding protein with a wash buffer to remove unbound material while maintaining binding of the guanine-nucleotide binding protein to the binding partner and inhibiting nucleotide hydrolysis and release; releasing the bound nucleotide from the guanine-nucleotide binding protein; and determining the ratio of guanine nucleotides released from the guanine nucleotide-binding proteins.
24. The method of claim 14 , further comprising washing the coated beads with the bound macromolecule with a wash buffer to remove unbound material while maintaining binding of the macromolecule to the capture reagent; and eluting the macromolecule from the capture reagent.