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System for rapid high-resolution gel electrophoresis

Inactive Publication Date: 2011-03-31
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012]In the presently described embodiments, electrophoretic systems, formulations, kits and methods are described that allow a user to perform electrophoresis experiments under conditions of high voltage and reduced run time. The systems formulations and methods improve upon those already known in the art and allow the user to generate electrophoretically resolved protein samples with a high degree of resolution and a reduced amount of time than currently available systems and formulations. An electrophoretic system, formulation or method practiced in accordance with the presently disclosed embodiments may be run, for example, at 50% higher field strength than comparable systems already in use in the art. In some embodiments, an electrophoresis gel prepared in accordance with the systems, methods and formulations described herein may be run at voltages above 225 V, above 250 V, above 275 V, above 300 V, above 325 V or above 350 V. In some embodiments, an electrophoresis gel prepared in accordance with the systems, methods and formulations described herein may be using a field strength in the range of about 12 v / cm to about 20 v / cm, or at least 12.0-13.0 V / cm, at least 13.5-14 V / cm, at least 15.0-15.5 V / cm, at least 16.0-17.0 V / cm, or at least 17.5-18.5 V / cm.

Problems solved by technology

However, maintaining uniformity between gels is difficult because each of these factors is sensitive to many variables in the chemistry of the gel system.

Method used

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  • System for rapid high-resolution gel electrophoresis
  • System for rapid high-resolution gel electrophoresis
  • System for rapid high-resolution gel electrophoresis

Examples

Experimental program
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Effect test

example 1

[0098]The first test gel formulation examined was an 8% SDS-PAGE resolving gel having the following composition: 100 mM Bis-Tris, 75 mM Tricine, 4 vol. % sucrose, and 20 mM BES. The stacking gel had the following composition: 357 mM Bis-Tris and 210 mM Chloride, pH 6.5 cast on top of the resolving gel. The S:R ratio of the gel was 1:4.

[0099]Five (5) μL of Mark12™ protein marker with Ponceau S tracking dye was loaded in each lane of the gel and the gel was run at 300 V for 15 minutes in NuPAGE® MES SDS Running Buffer (Invitrogen Corp., Carlsbad, Calif.). Completion of the gel run was determined based on the migration of Ponceau S tracking dye added separately to each sample to the bottom of the gel. At the completion of the run, the gel was stained with Coomassie® G-250 using SimplyBlue™ SafeStain (from Invitrogen Corp, Carlsbad, Calif.).

[0100]FIG. 1 shows the migration pattern of the marker bands resulting from this gel formulation. The 3.5 kDa insulin B band (indicated in FIG. 1) o...

example 2

[0101]An SDS-PAGE test gel was cast essentially as described in Example 1, except that the S:R ratio was changed to 41:59 in an attempt to improve the stacking and separation of bands migrating higher than the 55.4 kDa glutamic dehydrogenase band. Five (5) μL of Mark12™ protein marker was loaded in each lane of the gel and the gel was run at 300 V for 16 minutes in MES SDS Running Buffer. Completion of the gel run was determined based on the migration the Ponceau S tracking dye to the bottom of the gel and the gel was stained with SimplyBlue™ SafeStain as above.

[0102]The resulting gel (FIG. 2) shows the migration pattern of the marker bands resulting from this gel formulation. The 3.5 kDa Insulin B band migrated the entire length of the gel, although an additional 1 min of run time was required. The 200 kDa myosin band in the outermost lanes (i.e., lanes 1, 2, 9 and 10; indicated by the asterisk) was more diffuse than the corresponding band seen in lanes 3-8. Additionally, bands mig...

example 3

[0103]An SDS-PAGE test gel was cast essentially as described in Example 2, with the following changes: the 8% (8% T / 4.4% C) resolving gel was made using 100 mM Bis-Tris and 100 mM Tricine. The 4% (4% T / 3.8% C) stacking gel cast over the resolving gel had a pH of 6.1 and was made using 357 mM Bis-Tris and 150 mM sulfuric acid. In this example, sulfuric acid was used in order to provide a leading ion (SO4−2) that migrated slower than Cl−. The S:R ratio of the gel was 41:59. Five (5) μL of Mark12™ protein marker was loaded in each lane of the gel and the gel was run at 300 V for 18 minutes in MES SDS Running Buffer. Completion of the gel run was determined based on the migration of the Ponceau S tracking dye to the bottom of the gel after which the gel was stained with SimplyBlue™ SafeStain as above.

[0104]FIG. 3 shows the migration pattern of the marker bands resulting from this gel formulation. As shown, the use of sulfuric acid in the stacking gel yielded unsatisfactory results and a...

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Abstract

Electrophoretic systems, formulations and methods are described which allow a user to perform electrophoresis experiments under conditions of high voltage and with reduced run time. An electrophoretic system, formulation or method may be run at 50% higher field strength than comparable systems already in use in the art. The presently described systems and formulations may be run at voltages above 225 V, above 250 V, above 275 V, above 300 V, above 325 V or above 350 V. The time required for performing an electrophoresis experiment may be reduced to less than about 30 minutes, less than about 20 minutes, less than about 15 minutes or less than about 12 minutes.

Description

BACKGROUND[0001]1. Field of the Invention[0002]This invention relates to techniques and formulation for use in gel electrophoresis. More particularly, the present invention relates to novel systems and formulations for rapid, high resolution gel electrophoresis at substantially neutral pH.[0003]2. Description of Related Art[0004]Gel electrophoresis is a common procedure for the separation of biological molecules, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), polypeptides and proteins. In gel electrophoresis, the molecules are separated into bands according to the rate at which an imposed electric field causes them to migrate through a filtering gel.[0005]The basic apparatus used in this technique consists of a gel enclosed in a glass tube or sandwiched as a slab between glass or plastic plates. The gel has an open molecular network structure, defining pores which are saturated with an electrically conductive buffered solution of a salt. These pores through the gel are...

Claims

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Application Information

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IPC IPC(8): G01N27/447
CPCG01N33/561G01N27/44747
Inventor UPDYKE, TIMOTHYMILLER, JENNIFER
Owner LIFE TECH CORP
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