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Preparation method of standard protein sample and protein lysate

A protein lysing solution and lysing solution technology, which are applied in the preparation of test samples, measuring devices, instruments, etc., can solve the problems of high cost, inconvenience, tailing effect, etc., and achieve simple steps, convenient use and transportation, protein Clear banding effect

Inactive Publication Date: 2010-10-20
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In many cases, the specific thermal denaturation process is heating in a boiling water bath at 100°C for 30min, 10min, and 2min, or directly heating in a heating block at 100°C for 10min, 3min, or incubating at 37°C for 30min, etc. Standards for detecting protein quality in 1-DE and 2-DE proteome studies, but have varying degrees of tailing on 1-DE gel profiles
[0006] At present, the protein standard sample provided in the prior art, when used, the protein sample is dissolved in the buffer solution, but all need to be heated before use, and need to be transported at a low temperature of -20 degrees, which is inconvenient and expensive

Method used

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  • Preparation method of standard protein sample and protein lysate
  • Preparation method of standard protein sample and protein lysate
  • Preparation method of standard protein sample and protein lysate

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Using the BPP method to extract standard protein samples from Salicornia pratense roots, Salt mustard leaves, blood, cervical cancer Hela cells and Escherichia coli, and then put the above standard protein samples in protein lysis solution, the composition of the protein lysis solution is: 7 ~9M urea, 1~3M thiourea, 1~2% (v / v) of CHAPS (dimethylaminopropanesulfonic acid) and 12~15mM DTT (dithiothreitol), and then store the above mixed solution at room temperature, The mass volume concentration of the prepared protein standard sample is 1.0-5.0 μg / μl, and then added to one third of the volume of 3×Laemmli standard sample buffer when used. The 3×Laemmli standard sample buffer contains the following components: 625mM pH 6.8 Tris (trishydroxymethylaminomethane)-HCl, 2% (w / v) SDS (sodium dodecyl sulfate), 5% (v / v) β-mercaptoethanol, 10% (v / v ) glycerol and 0.001% (w / v) bromophenol blue, and directly conduct sodium dodecylsulfonate polyacrylamide SDS-PAGE gel electrophoresis ...

Embodiment 2

[0038] Mix the standard phosphorylase B, rabbit actin, soybean trypsin inhibitor and bovine serum albumin sample at a ratio of 1:1:1:2, and the mass volume concentration in the protein lysate is 1.0~ 5.0 μg / μl, and then dissolved in the lysate containing the following components: 7M urea, 2M thiourea, 2% CHAPS, 13mM DTT, and left at room temperature for 2 hours. Dispense 20 μl of each tube containing about 30 μg of protein, and then add to 10 μl 3×Laemmli standard sample buffer, 3×Laemmli standard sample buffer contains the following components: 625mM Tris (trishydroxymethylaminomethane)-HCl with a pH of 6.8, 2% (w / v) SDS (sodium dodecyl sulfate), 5% (v / v) β-mercaptoethanol, 10% (v / v) glycerol and 0.001% (w / v) bromophenol blue, PCR Heating in the instrument at 100°C for different times.

[0039] SDS-PAGE uses a 16 cm slab gel, the separating gel is 12.5% ​​polyacrylamide, and the stacking gel is 4% polyacrylamide, and then the above mixed solution is stored at room temperatur...

Embodiment 3

[0047] Take the protein extracted from the root of Salicornia salina by BPP method, and add the protein lysis solution according to its mass concentration in the protein lysis solution at 1.0-5.0 μg / μl. The composition of the protein lysis solution is: 7M urea, volume percentage 1.5% CHAPS and 14mM DTT, then store the above mixed solution at room temperature, and directly perform gel electrophoresis analysis without heating.

[0048] Refer to Example 1 for the process of using the BPP method to extract the protein from the roots of Salicornia.

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Abstract

The invention discloses a preparation method of a standard protein sample and protein lysate. The preparation method comprises the following steps: dissolving a standard protein sample into protein lysate with urea, and keeping the solution at the room temperature, thereby obtaining the standard protein sample which can be directly used for sodium dodecyl sulphate polyacrylamide gel electrophoresis SDS-PAGE without heating during loading for electrophoresis. The invention also discloses protein lysate applied in the preparation of the standard protein sample, which contains 7-9M of urea. The standard protein sample prepared in the method can be kept for a long time, no heating is needed, the operation is simple, and protein bands on the electrophoresis pattern are clear without obvious tailing.

Description

technical field [0001] The invention belongs to the field of proteomics, and in particular relates to a method for preparing a protein standard sample and a protein lysate used therefor. Background technique [0002] In 1970, Laemmli used sodium dodecylsulfonate polyacrylamide gel electrophoresis for the first time when separating the T4 phage coat protein, using different molecular weights of proteins to separate different proteins, which has a good separation effect. Since then, the technology has been widely used in biochemistry, forensic science, genetics and molecular biology research. Since the protein samples in SDS gel electrophoresis have the same charge per unit mass, the proteins bound to SDS are separated according to their molecular weights. At present, this method has become the most widely used biochemical analysis method in the world. According to Laemmli's method, protein samples were thoroughly mixed with SDS-PAGE loading buffer, boiled or heated for 1.5 m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N1/30G01N27/447
Inventor 王旭初卢秀丽田维敏郭安平
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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