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Method for preparing bidirectional electrophoresis cuttlefish ink sac complete protein sample

A two-dimensional electrophoresis and squid ink technology, which is applied in the preparation methods of peptides, the preparation of test samples, chemical instruments and methods, etc., can solve the problem of the lag in the research of the squid ink sac proteome, the interference of the two-dimensional electrophoresis process, and the discovery of the squid ink sac. The research of whole protein two-dimensional electrophoresis analysis and other problems, to achieve the effect of simple and easy method and great practical application value

Inactive Publication Date: 2011-11-23
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex composition of impurities in squid ink sacs, such as pigments, polysaccharides and nucleic acids, etc. can interfere with the two-dimensional electrophoresis process, there is no research report on the preparation of two-dimensional electrophoresis squid ink sac protein samples, so no two-dimensional electrophoresis of squid ink sac protein samples has been found. Studies of electrophoretic analysis, leading to a lag in squid ink sac-associated proteome studies

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] A preparation method for a two-dimensional electrophoresis squid ink sac full protein sample, comprising weighing 1 g of ink sac powder ground by liquid nitrogen and dissolving it in 10 ml of trichloroacetic acid and 0.02% trichloroacetic acid and 0.02% trichloroacetic acid pre-cooled at -20°C. In the acetone solution of dithiothreitol, let stand at -20°C for 1 hour, then centrifuge at 4°C and 10,000g to obtain the first precipitate; wash the above-mentioned first precipitate with alcohol with a mass concentration of 75% Finally, centrifuge again at 4°C and 10,000g to obtain a second precipitate; dry the above-mentioned second precipitate at 4°C, and then add 2ml of lysate, which contains urea with a concentration of 7mol / L, 2mol / L Thiourea, 40mmol / L tris, 6mmol / L dithiothreitol, 1mmol / L benzyl xanthyl fluoride, 1mmol / L disodium edetate, 40g / L 3 -[3-(cholamidopropyl) dimethylamino]propanesulfonic acid inner salt, 750ug / L leupeptin, 5ml / L ampholyte, let stand at 4°C for ...

Embodiment 2

[0014] A preparation method of two-dimensional electrophoresis cuttlefish ink sac full protein sample, which is basically the same as in Example 1, except that -20°C pre-cooled trichloroacetic acid containing 10% by mass concentration and 0.02% dithiothreo The acetone solution of sugar alcohol is 20ml, and the lysate is 4ml.

Embodiment 3

[0016] A preparation method of two-dimensional electrophoresis cuttlefish ink sac full protein sample, which is basically the same as in Example 1, except that -20°C pre-cooled trichloroacetic acid containing 10% by mass concentration and 0.02% dithiothreo The acetone solution of sugar alcohol is 15ml, and the lysate is 3ml.

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Abstract

The invention discloses a method for preparing a bidirectional electrophoresis cuttlefish ink sac complete protein sample, which comprises the following steps of: removing impurities such as pigment, polysaccharide, nucleic acid and the like through acetone solution to settle and separate ink sac powder complete protein, washing acetone by using alcohol, then cracking and extracting protein by using cracking solution, and diluting the protein by using hydration sampling buffer solution to obtain cuttlefish ink sac complete protein bidirectional electrophoresis sample solution, wherein every 300mu l of cuttlefish ink sac complete protein bidirectional electrophoresis sample solution contains 300 to 500mu g of cuttlefish ink sac complete protein. The method can effectively remove the impurities from the cuttlefish ink sac such as pigment, polysaccharide, nucleic acid and the like which interfere with a bidirectional electrophoresis process, create a condition for cuttlefish ink sac complete protein bidirectional electrophoresis and lay a foundation for cuttlefish ink sac related protein research; and the method is simple, feasible and quick, and has great practical application value.

Description

technical field [0001] The invention relates to a preparation method of two-dimensional electrophoresis protein samples, in particular to a preparation method of two-dimensional electrophoresis squid ink sac whole protein samples. Background technique [0002] Proteome analysis is an important technical means to study the changes of proteins in the ink sac and the corresponding surrounding environmental stimuli, which is conducive to in-depth understanding of its role in the development and function of the ink sac, and to achieve the purpose of controlling the occurrence of the ink sac. One of the key technologies to carry out the proteomics research of ink sac tissue is the preparation of protein samples in the ink sac tissue. The publication number is CN101260146, and the name is a patent application for the invention of a method of two-dimensional electrophoresis of Malus plant protein. Homogenize in acetone solution of β-mercaptoethanol, place at -20°C for 1 hour, then ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/14G01N1/34C07K14/435
Inventor 詹萍萍王春琳母昌考宋微微
Owner NINGBO UNIV
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