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Method for separating, enriching and identifying macromolecular weight protein

A high molecular weight, separation and enrichment technology, applied in the field of biochemical analysis, can solve the problems of difficult separation and analysis of large molecular weight proteins, and achieve the effects of good mass spectrometry compatibility, good reproducibility and large pore size

Inactive Publication Date: 2010-07-28
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The purpose of the present invention is to provide a method for separating, enriching and identifying large molecular weight proteins in view of the difficulty in the separation and analysis of large molecular weight proteins in the prior art

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  • Method for separating, enriching and identifying macromolecular weight protein
  • Method for separating, enriching and identifying macromolecular weight protein
  • Method for separating, enriching and identifying macromolecular weight protein

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 Research on the Optimum Separation Molecular Weight Range of Protein High Molecular Weight End in Low Melting Point Agarose / Polyacrylamide Mixed Gel

[0034] Standard molecular weight marker proteins (molecular weight range 10-250kDa) were used as standard samples to investigate the separation molecular weight ranges of low melting point agarose and polyacrylamide gel mixtures in different proportions. figure 1 Gel electrophoresis results showing increasing ratio of 0.8% lmpAg from 0 to 1 / 2. As the proportion of lmpAg increases, the average pore size of the hybrid gel increases, and the molecular weight range that can be separated tends to be higher. The separation molecular weight range of each gel can be calculated by Ferguson's law, such as figure 2 Shown: 7% SDS-PAGE, 35-270kDa; SDS-lmpAgPAGE 1 (0.8%SDS-lmpAg / 7%SDS-PAGE gel: 1 / 3, v / v), 60-330kDa; SDS-lmpAgPAGE 2 (0.8 %SDS-lmpAg / 7%SDS-PAGE gel: 1 / 2, v / v), 75-360kDa; SDS-lmpAgPAGE 3 (0.8%SDS-lmpAg / 7%SDS-P...

Embodiment 2

[0035] Example 2 Application example of AgPAGE-LC-ESI-MS / MS route

[0036] The SD-rat brain tissue protein extract purified by GST-Pulldown was used for one-dimensional mixed gel electrophoresis, and compared with the conventional low concentration polyacrylamide gel. image 3 It shows that the use of lmpAgPAGE as the electrophoresis medium makes the separation effect of high molecular weight end significantly better than that of ordinary gel, and the Spectrin isoforms are well separated. The mass spectrometry identification supports this result, see Table 2.

[0037] Table 2 is a list of ESI-MS / MS identification results of contractrin isoforms (Spectrin isoforms). The results showed that the contractile protein isomers were derived from Sprague-Dawley rat (SD-rat) brain purified by glutathione-S-transferase affinity chromatography test (GST-Pulldown). Tissue protein extract. The purified brain tissue protein extract was first separated by low-melting point agarose and polya...

Embodiment 3

[0040] Example 3 Application example of IEF-AgPAGE-MALDI-TOF MS / MS route

[0041] The whole protein of SD-rat large intestine tissue was used for two-dimensional gel electrophoresis. The first dimension used solid-phase gradient Ph dry strip isoelectric focusing, and the second dimension used hybrid gel or ordinary low-concentration polyacrylamide gel electrophoresis. glue, such as Figure 4 shown. ImageMaster 2-D software analysis showed that the number of proteins with a molecular weight higher than 100kDa using the mixed gel was significantly higher than that using the ordinary gel, which were 298 protein spots and 224 protein spots, respectively. For the optional 26 points on the gel, two enrichment methods were used: nano-core-shell complex enrichment and desalination method and centrifugal freeze-drying method. The mass spectrometry data showed that the identification rate of these 26 spots reached 100% by the material enrichment method, while only 7 protein spots were...

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Abstract

The invention relates to a method for separating, enriching and identifying macromolecular weight protein, belonging to the field of biochemical analysis. In the invention, a mixed gel of low-melting point agarose and polyacrylamide is used as an electrophoresis supporting medium, and an analysis path of gel slab electrophoresis combined with efficient liquid-phase chromatography - electrical spraying mass spectrum identification and another analysis path of solid-phase gradient pH dry adhesive tape isoelectro-focusing combined with gel slab electrophoresis separation - matrix auxiliary laser desorption ionization mass spectrum identification are collectively used to separate and identify the macromolecular weight protein. The gel provided by the invention has the advantages of big aperture, high mechanical strength, high resolution ratio, good repeatability and good mass spectrum compatibility; the macromolecular weight separation scope can be adjusted based on different component proportions; and protein with the molecular weight interval above 100kDa can be selectively separated with repeatability RSD up to 7.6%, thereby obviously improving the identification ratio. The method is an effective tool for separating the macromolecular weight protein, which can be applied to proteomics study field.

Description

technical field [0001] The invention belongs to the field of biochemical analysis, and relates to a method for separating, enriching and identifying large molecular weight proteins. In particular, it relates to a method for separating large-molecular-weight proteins by using low melting point agarose and polyacrylamide mixed gel (AgPAGE) as an electrophoresis support medium. Background technique [0002] Proteome research is a powerful tool for discovering biological targets of clinically significant diseases. At present, proteome research is increasingly being paid close attention to by researchers at home and abroad. Time has shown that in order to reduce the complexity of real samples, the separation of proteins is crucial. The prior art discloses that large-scale protein separations mainly rely on gel electrophoresis and liquid chromatography techniques. The former is still the first choice for the separation of complex protein samples due to its mature technology and...

Claims

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Application Information

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IPC IPC(8): G01N30/72G01N1/28G01N1/30
Inventor 沈雯雯陆豪杰杨芃原
Owner FUDAN UNIV
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