Compositions and methods for improving resolution of biomolecules separated on polyacrylamide gels

• US 9,057,694 B2
• Filed: 06/26/2013
• Issued: 06/16/2015
• Est. Priority Date: 12/29/2005
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Updyke, Timothy, Beardslee, Thomas
• Primary Examiner(s): Noguerola, Alex
• Application Number:
  US13/928,137
• Publication Number:
  US 20140034500A1
• Time in Patent Office: 720 Days
• Field of Search: 204450-470, 204600-616
• US Class Current: 1/1
• CPC Class Codes: C07K 1/26 Electrophoresis
• CPC Class Codes: C08J 2333/26 Homopolymers or copolymers of acrylamide or methacrylamide
• CPC Class Codes: C08J 3/075 Macromolecular gels
• CPC Class Codes: C08K 5/0008 Organic ingredients according to more than one of the "one dot" groups of C08K5/01 - C08K5/59
• CPC Class Codes: C08L 33/26 Homopolymers or copolymers of acrylamide or methacrylamide
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture
• CPC Class Codes: G01N 27/44756 Apparatus specially adapted therefor

Abstract

Gels, such as polyacrylamide gels, are provided that include linear polyacrylamide in the stacking gel. Native gels that include linear polyacrylamide in the stacker can be used to separate biomolecular complexes, such as protein complexes. Gel cassettes in which the gap width between front and back plates does not vary by more than 5% at the upper edge of the cassette are also provided. The gel cassettes can be used for electrophoretic separation of proteins and protein complexes on native gels, such as native gels that include linear polyacrylamide in the stacker. The native gels can have multiple wells for electrophoresing at least one sample and/or at least one molecular weight standard.

Claims

1. A polyacrylamide gel for the separation of biomolecules, comprising a stacking gel and a separating gel, wherein the stacking gel comprises linear, non-crosslinked polyacrylamide at a concentration of from 0.005% to 1%, and wherein the separating gel does not comprise linear, non-crosslinked polyacrylamide.

2. The polyacrylamide gel of claim 1, wherein the polyacrylamide and bisacrylamide concentration of the stacking gel is between 2% and 6%, and is optionally between 2.5% and 5%.

3. The polyacrylamide gel of claim 2, wherein the concentration of the linear, non-crosslinked polyacrylamide of the stacking gel is from 0.01% to 0.5%, and optionally from 0.02% to 0.1%.

4. The polyacrylamide gel of any of claim 1, wherein the polyacrylamide gel comprises SDS (sodium dodecyl sulfate).

5. The polyacrylamide gel of any of claim 1, wherein the polyacrylamide gel is a non-denaturing gel, wherein optionally the non-denaturing gel is a gradient gel or a Blue Native Gel.

6. A cassette for performing native gel electrophoresis, wherein the cassette contains a polyacrylamide gel of according to claim 1 and has a consistent gap width across its upper edge.

7. The cassette of claim 6, wherein the cassette is plastic.

8. The cassette of claim 6, wherein the cassette is made by attaching a front plate to a back plate by one or more of the following:

being moulded as a single piece along with edge pieces that connect the front and back plates at the side;

being welded together;

being fastened together with adhesives;

being thermally treated;

or being held with attachment means screws, pins, snaps, or clamps.

9. The cassette of claim 6, wherein the cassette has a gap width across its upper edge in the range from 0.1 millimeters to 5 millimeters.

10. The cassette of claim 6, wherein the gap width varies by less than 5%, preferably by less than 2%.

11. A method of separating biomolecules on an electrophoresis gel, comprising:

applying one or more samples comprising one or more biomolecules to a polyacrylamide gel of according to claim 1; and

electrophoretically separating one or more biomolecules on the gel.

12. The method of claim 11, wherein the gel is a multiwell gel.

13. The method of claim 11, further comprising applying one or more molecular weight marker sets to the electrophoresis gel, optionally further comprising estimating or calculating the molecular weight of one or more biomolecules or biomolecular complexes on the electrophoresis gel.

14. The method of claim 11, further comprising staining the gel.

15. The method of claim 11, wherein the polyacrylamide gel is within a cassette that has a consistent gap width across its upper edge.