Preparation of defect-free polyacrylamide electrophoresis gels in plastic cassettes

a technology of polyacrylamide and electrophoresis gel, which is applied in the field of polyacrylamide gels, can solve the problems of apparent solute band distortion and band distortion, and achieve the effect of uniform pore siz

Inactive Publication Date: 2005-03-17
BIO RAD LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention resides in the discovery that the interface irregularities of polyacrylamide gels that are precast in plastic gel cassettes can be reduced or eliminated by the inclusion of an oxygen scavenger in the gel-forming solution from which the gel is cast. The monomer mixture in the solution is polymerized with the scavenger present in the solution, and the result is a pre-cast gel with a substantially uniform pore size throughout. Band resolution in the cassette is then comparable to the band resolution that can be obtained with polyacrylamide gels in glass enclosures.

Problems solved by technology

The plates are typically made of plastic, and a difficulty that has been observed is an apparent distortion of the solute bands due to irregularities in the gel pore size near the interface between the gel and the plastic.
These irregularities are detrimental to the electrophoretic analysis since the pore size affects the migration of the solute bands, and pore size variations cause distortions of the bands.
The problem is particularly acute with polyacrylamide gels formed in plastic enclosures.

Method used

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  • Preparation of defect-free polyacrylamide electrophoresis gels in plastic cassettes

Examples

Experimental program
Comparison scheme
Effect test

example 1

A gel-forming solution was prepared by combining the following components in an aqueous solution in the concentrations indicated: acrylamide / N,N′-methylene-bisacrylamide (T 12%, C=2.67%) 0.375 M tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), pH 8.6 0.067% TEMED 0.05% ammonium persulfate 6 mM sodium sulfite

The acrylamide / bisacrylamide mixture in the solution was allowed to polymerize at room temperature (approximately 20° C.) for 30-60 minutes to form a gel ready for electrophoresis.

example 2

The following solutions were prepared for use in forming resolving and stacking gels for a gel cassette constructed with one glass plate and one acrylic plastic plate. All percents are by weight unless otherwise indicated, and all solutions are aqueous solutions.

TABLE IResolving Gel:deionized H2O34.5mL1.5 M Tris-HCl, pH 8.825.0mL30% (37.5:1)40.0mLacrylamide / bisacrylamide10% ammonium persulfate500μLTEMED50μLStacking Gel:deionized H2O6.2mL1.5 M Tris-HCl, pH 6.82.5mL30% (37.5:1)1.3mLacrylamide / bisacrylamide10% ammonium persulfate50μLTEMED10μL

A stock solution of sodium sulfite (1 M aqueous) was also prepared, and added to the resolving and stacking gel solutions in different levels as follows: (1) No sodium sulfite added to either gel (2) 57.1 μL of stock solution added to both gel solutions to a final sodium sulfite concentration of 72.02 μg / mL (0.571 mM) in each solution (3) 571.4 μL of stock solution added to both gel solutions to a final sodium sulfite concentration of 720.2 ...

example 3

The following solutions were prepared for use as resolving and stacking gels for a gel cassette formed from two plates of uncoated polystyrene-acrylonitrile copolymer (SAN 880B TYRIL®, The Dow Chemical Company, Midland, Mich., USA). All percents are by weight unless otherwise indicated, and all solutions are aqueous solutions.

TABLE IIResolving Gel:deionized H2O34.5mL1.5 M Tris-HCl, pH 8.825.0mL30% (37.5:1)40.0mLacrylamide / bisacrylamide10% ammonium persulfate500μLTEMED50μLStacking Gel:deionized H2O6.2mL1.5 M Tris-HCl, pH 6.82.5mL30% (37.5:1)1.3mLacrylamide / bisacrylamide10% ammonium persulfate50μLTEMED10μL

A stock solution of 1 M aqueous sodium sulfite was also prepared and added to the resolving and stacking gel solutions in different levels as follows: (1) No sodium sulfite added to either gel (2) 57.1 μL of stock solution added to both gel solutions to a final sodium sulfite concentration of 72.02 μg / mL (0.571 mM) in each solution (3) 571.4 μL of stock solution added to both ...

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Abstract

Irregularities in the pore structures of polyacrylamide gels that are formed in cassettes against plastic walls are reduced or eliminated by the inclusion of an oxygen scavenger in the gel-forming solution. Avoidance of the irregularities results in electropherograms with fewer distortions in the solute bands.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to polyacrylamide gels as used in slab gel electrophoresis. 2. Description of the Prior Art Slab gels are particularly useful for electrophoresis in view of their ability to accommodate multiple sample analyses and the ease with which the electropherograms can be observed and read by simply identifying the locations of the various bands on the gels that correspond to the individual components. Polyacrylamide is a gel material that is widely used in slab gels. Slab gels are frequently supplied in pre-cast form, retained between two flat transparent plates in a cassette. The plates are typically made of plastic, and a difficulty that has been observed is an apparent distortion of the solute bands due to irregularities in the gel pore size near the interface between the gel and the plastic. These irregularities are detrimental to the electrophoretic analysis since the pore size affects the migration of th...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C08F2/10C08F4/00C08F2/38C08F4/40C08F20/56C08F220/56C08K5/17G01N27/447
CPCC08F220/56C08K5/17G01N27/44704Y10S526/934G01N27/44747Y10S524/916C08L33/26
Inventor PANATTONI, CORY M.
Owner BIO RAD LAB INC
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