Subcellular western blotting of single cells
Electrophoretic separation methods and systems for performing the same are provided. The methods and systems find use in a variety of different electrophoretic separation applications, such as sub-cellular Western blotting of single cells.
1. A method of detecting an analyte, the method comprising:
(a) contacting a sample comprising a cell with a polymeric separation medium comprising a microwell, wherein the polymeric separation medium comprises functional groups that covalently bond one or more cellular components to the polymeric separation medium upon application of an applied stimulus;
(b) contacting the polymeric separation medium with a first buffer sufficient to differentially lyse a first sub-cellular compartment of the cell to produce a first set of cellular components;
(c) applying an electric field to the polymeric separation medium in a manner sufficient to move at least some of the first set of cellular components into the polymeric separation medium to produce a first set of separated cellular components in the polymeric separation medium;
(d) immobilizing the first set of separated cellular components in the polymeric separation medium; and
(e) contacting the polymeric separation medium with a second buffer sufficient to differentially lyse a second sub-cellular compartment of the cell to produce a second set of cellular components.
2. The method of claim 1 , wherein the contacting (a) is sufficient to position the cell in the microwell.
3. The method of claim 1 , wherein the contacting (b) comprises contacting a surface of the polymeric separation medium with a hydrogel layer comprising the first buffer.
4. The method of claim 1 , wherein the first buffer is sufficient for the contacting (b) and the applying (c).
5. The method of claim 1 , wherein the immobilizing comprises covalently bonding the first set of separated cellular components to the polymeric separation medium.
6. The method of claim 1 , further comprising applying an electric field to the polymeric separation medium in a manner sufficient to move at least some of the second set of cellular components into the polymeric separation medium to produce a second set of separated cellular components in the polymeric separation medium.
7. The method of claim 6 , wherein the second set of cellular components is separated in a direction different from the first set of cellular components.
8. The method of claim 6 , further comprising immobilizing the second set of separated cellular components in the polymeric separation medium.
9. The method of claim 1 , further comprising detecting the separated cellular components.
10. The method of claim 9 , wherein the detecting comprises contacting the separated cellular components with an analyte detection reagent.
11. The method of claim 10 , further comprising contacting the separated cellular components with one or more secondary reagents.
12. The method of claim 1 , further comprising imaging the polymeric separation medium to produce an image of the separated cellular components.
13. The method of claim 1 , wherein the functional groups are co-polymerized with the polymeric separation medium.
14. A system for detecting an analyte, the system comprising:
a polymeric separation medium comprising a microwell, wherein the polymeric separation medium comprises functional groups that covalently bond one or more cellular components to the polymeric separation medium upon application of an applied stimulus;
a first hydrogel layer comprising a first buffer sufficient to differentially lyse a first sub-cellular compartment of a cell to produce a first set of cellular components in the microwell; and
a second hydrogel layer comprising a second buffer sufficient to differentially lyse a second sub-cellular compartment of the cell to produce a second set of cellular components in the microwell.
15. The system of claim 14 , wherein the first hydrogel layer is positioned on a surface of the polymeric separation medium such that the first buffer is in fluid communication with the microwell.
16. The system of claim 14 , wherein the microwell is dimensioned to accommodate a single cell.
17. The system of claim 14 , wherein the polymeric separation medium comprises an array of microwells in the polymeric separation medium.
18. The system of claim 14 , wherein the polymeric separation medium comprises 100 or more microwells.
19. A kit comprising:
a polymeric separation medium comprising a microwell, wherein the polymeric separation medium comprises functional groups that covalently bond one or more cellular components to the polymeric separation medium upon application of an applied stimulus:
a hydrogel;
a first buffer sufficient to differentially lyse a first sub-cellular compartment of a cell to produce a first set of cellular components in the microwell;
a second buffer sufficient to differentially lyse a second sub-cellular compartment of the cell to produce a second set of cellular components in the microwell; and
a packaging containing the system.