Electrophoretic matrix staining composition and method

Patent metadata

Field	Value
Publication number	US-9709527-B2
Application number	US-201414494754-A
Country	US
Kind code	B2
Filing date	Sep 24, 2014
Priority date	Sep 26, 2013
Publication date	Jul 18, 2017
Grant date	Jul 18, 2017

Abstract

Staining compositions and methods for use with a matrix, such as an electrophoretic gel, containing separated biopolymers. The compositions including an acid, an organic solvent, and a generally planar, fluid-permeable gel contact sheet consisting primarily of a non-cellulosic material. The acid and organic solvent may be sorbed to the fluid-permeable gel contact sheet, or may be sorbed to a layers contactable with a non-gel contact side of the gel contact sheet. In one embodiment, the composition is a source electrophoretic stain composition including a staining reagent. In one embodiment, the composition is a sink electrophoretic stain composition including a sorbent.

First claim

What is claimed is:

1. A method for electrophoretically staining biopolymers embedded in a matrix, the method comprising
contacting a first side of the matrix containing the biopolymer with a non-cellulosic first matrix contact sheet or a first matrix contact sheet including a blend containing cellulosic material, the first matrix contact sheet having a matrix contact surface and a stain reagent,
contacting a second side of the matrix containing the biopolymer with a second matrix contact sheet comprising a non- or partially-cellulosic material, having a matrix contact surface and comprising a destain solution comprising an organic solvent, an acid, water, and optionally a buffering agent, the contacting of the first matrix contact sheet and the second matrix contact sheet with the matrix forming a multi-layered assembly having a thickness, and
applying an electrical force across the thickness of the assembly sufficient to move the stain reagent into the matrix, thereby staining the biopolymer in the matrix, and to move excess free stain reagent out of the matrix, thereby destaining the matrix.

2. The method of claim 1 where the first matrix contact sheet further comprises at least one additional sheet distal from the first side of the matrix and/or the second matrix contact sheet further comprises at least one additional sheet distal from the second side of the matrix, the at least one additional sheet being the same or different from the first matrix contact sheet and/or the second matrix contact sheet.

3. The method of claim 1 where the first matrix contact sheet is capable of absorbing at least 5 ml of a staining solution, but less than 15 ml of the staining solution; a thickness of the first matrix contact sheet is less than 0.5 inches; and/or the matrix contact surface is sufficient to provide maximum contact between the first matrix contact sheet and the matrix.

4. The method of claim 1 where the first matrix contact sheet or the first and the second matrix contact sheet is polyester, polyester/cellulose blend, polypropylene, polypropylene/cellulose blend, polyacrylamide, nitrocellulose, non-cellulose, polysaccharide, agarose, felt, polyvinylidene fluoride (PVDF), polyvinylidene difluoride, fine woven cotton, wool, regenerated cellulose fiber, Lyocell, and combinations thereof.

5. The method of claim 4 where the first matrix contact sheet is polyester, polyester/cellulose blend, polypropylene, polypropylene/cellulose blend, polyacrylamide, nitrocellulose, non-cellulose, polysaccharide, agarose, felt, polyvinylidene fluoride (PVDF), polyvinylidene difluoride, regenerated cellulose fiber, Lyocell, and combinations thereof.

6. The method of claim 2 where the at least one additional sheet is polyester, polyester/cellulose blend, polyacrylamide, nitrocellulose, polysaccharide, agarose, felt, polyvinylidene fluoride (PVDF), polyvinylidene difluoride, a fabric, a foam, a sponge, and combinations thereof.

7. The method of claim 2 where a stain solution and/or the destain solution is present in the at least one additional sheet which is not in contact with the matrix.

8. The method of claim 1 where the second matrix contact sheet comprises a sorbent selected from the group consisting of wound dressing non-adhesive material, woven and non-woven cotton, foam, felt, agarose, agarose/polyacrylamide matrix, alumina, silica, polysaccharides, polymer, and combinations thereof.

9. The method of claim 1 further comprising, prior to contacting the matrix with the first and second matrix contacting sheet, contacting the first matrix contacting sheet with a stain solution, such that the stain solution is absorbed into the first matrix contact sheet, and contacting the second matrix contacting sheet with the destain solution, such that the destain solution is absorbed into the second matrix contact sheet.

10. The method of claim 1 further comprising a staining solution, where the staining solution comprises an acid, an organic solvent, and the staining reagent.

11. The method of claim 10 where the acid is acetic acid and the organic solvent is ethanol or isopropanol.

12. The method of claim 10 where the staining solution further comprises a buffering agent.

13. The method of claim 12 where the buffering agent is sodium phosphate.

14. The method of claim 1 where the staining reagent is a protein stain, and is selected from the group consisting of Coomassie Blue R-250, Coomassie Blue G-250, silver stain, hydrophobic or charged staining reagents, neutral staining reagents, and protein modification stains.

15. The method of claim 14 where the charged staining reagents include chromogenic, fluorescent, chemiluminescent, and/or bioluminescent molecules.

16. The method of claim 14 where the neutral staining reagent further comprises a non-charged organic or non-organic solvent.

17. The method of claim 14 where the protein modification is at least one of phosphorylation, glycosylation, acetylation, nitrosylation, SUMOylation, or epitope tags.

18. The method of claim 1 further comprising a stain solution, where the stain solution comprises 25% ethanol, 5% acetic acid, and 0.01% Coomassie Brilliant Blue dye R-250 or G-250, and the destain solution comprises 25% ethanol and 5% acetic acid.

19. The method of claim 1 where the matrix is selected from the group consisting of SDS-polyacrylamide gels, native polyacrylamide gels, membranes, and filters.

20. A method for electrophoretically staining biopolymers embedded in a matrix, the method comprising
forming an electrophoretic, multi-layered assembly by
contacting a first side of the matrix containing the biopolymer with a first matrix contact sheet consisting primarily of a non-cellulosic or partially cellulosic material having a matrix contact side in direct contact with the first side of the matrix, the first matrix contact sheet comprising a stain solution comprising a stain reagent and a solvent,
contacting a second side of the matrix containing the biopolymer with a second matrix contact sheet comprising a non- or partially-cellulosic material having a matrix contact side in direct contact with the second side of the matrix, the second matrix contact sheet comprising a destain solution comprising an organic solvent, an acid, water, and optionally a buffering agent,

positioning the assembly between a first electrode and a second electrode and in electrical contact with said electrodes; and
applying a current to the electrodes and across the assembly to provide an electric force sufficient to move the staining reagent into the matrix, thereby staining the biopolymer in the matrix, and to move excess free staining reagent out of the matrix, thereby destaining the matrix.

21. The method of claim 20 where the first electrode is a cathode and is stainless steel or titanium and the second electrode is an anode and is platinum-coated titanium or carbon.

Assignees

• Pierce Biotechnology Inc

Inventors

• Dworecki Boguslawa R
• Desai Surbhi
• Kilmer Gregory John
• Bell Peter Alan
• Webb Brian Lynn

Classifications

• G01N2001/302

  Stain compositions · CPC title

• G01N27/44726 Primary

  using specific dyes, markers or binding molecules · CPC title

• G01N27/44739

  Collecting the separated zones, e.g. blotting to a membrane or punching of gel spots · CPC title

• G01N1/30

  Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis} · CPC title