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Cumulative time-resolved emission two-dimensional gel electrophoresis

A technology of two-dimensional gel electrophoresis and fluorescent dyes, which is applied to the analysis of materials, material analysis through electromagnetic means, material analysis through optical means, etc., and can solve problems that prevent the use and development of TRF

Active Publication Date: 2011-04-27
HIKARI BIO AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current lack of this feature in modern 2-DGE image acquisition equipment prevents the use and development of TRF in 2-DGE

Method used

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  • Cumulative time-resolved emission two-dimensional gel electrophoresis
  • Cumulative time-resolved emission two-dimensional gel electrophoresis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057]In this example, proteins were first extracted from rat airways by lysis lavage (14) in 2-DGE compatible urea-based lysis buffer (7M urea, 2M thiourea, 4% w / v Rapid solubilization of the airway epithelial proteome in CHAPS, 0.5% Triton-X 100, 2% v / v protease inhibitor cocktail). Protein concentrations were determined using the Bradford method, and proteins were separated using 2-DGE. An aliquot of 400 μg protein / sample was diluted to 350 μl with lysis buffer and IPG buffer of the appropriate pH range was added to a final concentration of 1% v / v. By rehydrating overnight at room temperature, protein samples were loaded onto IPG gel strips (GEHealthcare) and passed through the following gradient protocol at 20°C using a Multiphor II Electrophoresis unit electrophoresis instrument and an EPS3501XL power supply electrophoresis tank: 0-50V , 1min.; 50V, 1 hr; 50-1000V, 3 hr; 1000-3500V, 3hr; 3500V, 19hr (total 74.9kVh) for isoelectric focusing (IEF). After IEF, the strips w...

Embodiment 2

[0063] Macrophages extracted by bronchoalveolar lavage from smoking and never-smoking human subjects were lysed with 2-DGE lysis buffer (see Example 1). Internal standards were generated by pooling equal amounts of protein from all subjects included in the study and were labeled with NHS ester-conjugated Cy2 (minimum DIGE reagent) according to the manufacturer's recommendations (GE Healthcare, Uppsala, Sweden). Protein samples from smoking and never-smoking subjects were randomly divided into two groups and labeled with Cy3 and Cy5 (minimal DIGE reagent), respectively. Cy2-labeled internal standards were co-separated on 2-DGE gels using one Cy3 and one Cy5-labeled sample according to the protocol described in Example 1. Three different protein markers were visualized in an automated sequence of three different scanning protocols using a combination of spectral and time-resolved fluorescence. The specific operation is as follows:

[0064] A) Cy2 fluorescent dye (internal stan...

Embodiment 3

[0069] As an additional step to the protocol described in Example 2, perform SYPRO TM Ruby staining facilitates quantification of total protein content in DIGE gels. In prior art, due to spectral overlap, especially in SYPRO TM Spectral overlap in the emission curves of Ruby, Cy3 and Cy5 makes it impossible to use DIGE and SYPRO in the same 2-DGE gel TM Ruby fluorescent dye. Embodiments of the present invention utilize temporal dimensions to facilitate the incorporation of these methods with standard protocols (as exemplified in FIG. 3 ). After completing the steps outlined in Example 2, following steps B and C outlined in Example 1, in SYPRO TM Total protein content was quantified after Ruby staining.

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Abstract

A new instrumental design is provided for in-gel detection and quantification of proteins. This new platform, called Cumulative Time-resolved Emission 2-Dimensional Gel Electrophoresis, utilizes differences in fluorescent lifetime imaging to differentiate between fluorescence from specific protein labels and non-specific background fluorescence, resulting in a drastic improvement in both sensitivity and dynamic range compared to existing technology. The platform is primarily for image acquisition of two-dimensional gel electrophoresis, but is also applicable to protein detection in one-dimensional gel systems as well as proteins electroblotted to e.g. PVDF membranes. Given the increase in sensitivity of detection and dynamic range of up to 5-6 orders of magnitude compared to existing approaches, the described invention represents a technological leap in the detection of low abundance cellular proteins, which is desperately needed in the field of biomarker discovery.

Description

technical field [0001] The present invention relates to a method for in-gel detection and quantification of proteins and a new image acquisition technique. This new platform, called Cumulative Time-Resolved Emission Two-Dimensional Gel Electrophoresis (CuTEDGE), exploits differences in fluorescence lifetimes to distinguish fluorescence from specific protein markers from nonspecific background fluorescence, resulting in improvements in sensitivity and dynamics compared to existing techniques. The range has been significantly improved. Background technique [0002] "Proteomics" refers to the study of the totality of proteins in the genome (proteome), a term proposed by Marc Wilkins in 1994. Over the past decade, methods for the simultaneous quantification of thousands of proteins within cells or tissues have been developed and used eg for biomarker discovery or mechanistic studies of cellular processes. Two-dimensional gel electrophoresis (2-DGE) was the first method suitabl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447
CPCG01N27/44773G01N27/44778G01N2021/6413G01N21/6408G01N27/44721G01N21/6428G01N2021/6439G01N27/44726
Inventor 奥萨·惠洛克
Owner HIKARI BIO AB
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