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Method for producing western blot resin with dual recognition group polymer chain and application thereof

A western blotting and dual recognition technology, applied in the field of separation and enrichment of natural trace proteins, can solve the problems of single imprinted protein method, limit the practical significance of the application of western blotting polymers, etc. The effect of improving specificity

Inactive Publication Date: 2010-02-03
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is a single method for imprinting proteins, monomer polymerization occurs in organic solvents, and the types of proteins used for imprinting are few and common, with a large amount in the organism or easily obtained, which limits the ability of western imprinting polymers. The practical significance of the application

Method used

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  • Method for producing western blot resin with dual recognition group polymer chain and application thereof
  • Method for producing western blot resin with dual recognition group polymer chain and application thereof
  • Method for producing western blot resin with dual recognition group polymer chain and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0024] First, the preparation of the auxiliary recognition chain: take 2.3g of azobisisobutyronitrile (AIBN) as the initiator, 0.5ml of 4-vinylpyridine and 30ml of acrylonitrile (AN) as the reaction monomer, and 200ml of absolute ethanol as the solvent , and sequentially added to a pre-dried three-necked round-bottomed flask, and the molar ratio of acrylonitrile and 4-vinylpyridine was 1:99. The mass ratio of the reaction monomer acrylonitrile and the initiator azobisisobutyronitrile is 10.6:1. Nitrogen protection, electromagnetic stirring, 80 ° C water bath heating, reflux condensation, reaction 8hr. The product was poured into absolute ethanol to precipitate while it was hot, washed repeatedly, and dried in vacuum at 50° C. for 24 hours to obtain the random copolymer PANVP. Take 2 g of the refined random copolymer of acrylonitrile and 4-vinylpyridine (PANVP), add it to 15 ml of 85% (w / w) concentrated sulfuric acid, and hydrolyze it at 100° C. for 4 hours, so that the nitril...

Embodiment 2

[0027] Example 2, Application of Western Blot Resin in Protein Separation

[0028] At 4°C, the above molecularly imprinted resin was mixed with 2 mL of porcine hepatocyte extract (cytosol), and the resin after 15 hours of spin adsorption was washed with 10 mM Tris-HCl (pH 8.0) buffer solution for 5 times, 2 mL each time, and then Wash twice with 2 mL buffer solution containing 10mM Tris-HCl (pH 8.0) and 100mM KCl, wash three times with buffer solution containing 10mM Tris-HCl (pH 8.0) and 300mM KCl, ) and a buffer solution of 500mM potassium chloride to wash the resin twice to obtain a washing solution. The Bradford method was used to detect the washing solution of potassium chloride solution and the pig liver cell extract solution to obtain the total protein content; then take 800 μL of the washing solution, and use the trichloroacetic acid / sodium deoxycholate (TCA / DOC) method to precipitate protein, protein After liquid sample preparation, constant flow sodium dodecylsulfon...

Embodiment 3

[0037] Embodiment 3, the preparation method of cloned protein porcine cyclophilin 18:

[0038] 1. Cloning of target gene

[0039] In this experiment, pig liver mRNA was used as a template for reverse transcription PCR (RT-PCR), and the PCR product and plasmid vector pGEX-5X1 were modified with restriction endonucleases. After ligation, they were transformed into E. coli DH5α and the plasmid was identified. .

[0040] (1) In a 200mL thin-walled PCR tube, add the required components in the following order for PCR reaction: 1 μL of RNase inhibitor, 100 ng of pig liver cell RNA, 25 μL of reaction buffer, and 10 pmol of forward primer. Reverse primer 10pmol, Taq mixed enzyme 1μL, double distilled water 21μL.

[0041] (2) Weigh 0.75g agarose, add 50mL 1×TAE, mix well and heat to boiling, take it out and add 2μL ethidium bromide (EB) solution (10mg / mL) when it cools slightly, pour the glue into Insert the comb into the gel stage of the electrophoresis tank; take out 16 μL of the s...

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Abstract

The invention relates to a preparation method of protein blot resin with a dual recognition group polymer chain and the application, and in order to improve the specificity of identifying objective protein, the invention brings in the assistant recognition chain with a random dual recognition group for limiting length. The random copolymer of acrylonitrile and 4-ethylene pyridine is used as the frame of the assistant recognition chain, and the recognition group is carboxyl and pyridine group. Then 80-100 objects polyvinyl alcohol resin microballoon sphere is chosen as the carrier of the assistant recognition chain. According to the recognition requirement of protein, the covering proportion of recognition group in short chain is set to 10 percent respectively. The invention uses cloned pigCyP 18(pCyP18) protein as a moulding board. The invention has multi-dimensional and multi-angle recognition to protein, increases recognition position points, improves the recognition specificity, and can better have special recognition of a plurality of high concentration collection of objective protein. The absorbing quantity to the objective protein is 600ng / g, and the content in the total protein is improved by more than 200 times.

Description

【Technical field】: [0001] The invention belongs to the technical field of synthesis and application of protein imprinting polymers, in particular to the synthesis and application of protein imprinting resins with dual recognition groups, using cloned proteins as templates to synthesize protein imprints with limited-length polymer chains for auxiliary recognition Polymers are specifically applied to the separation and enrichment of natural trace proteins. 【Background technique】: [0002] Molecular imprinting technology is the specific recognition ability at the molecular level that polymers possess by "memorizing" the three-dimensional space of imprinted molecules. Due to the particularity of protein structure and the requirement of biological activity, imprinted protein has become a research hotspot in the field of molecular imprinting in recent years. Using non-covalent interactions is the easiest way to imprint proteins. Commonly used weak intermolecular forces include: ...

Claims

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Application Information

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IPC IPC(8): C08F261/04C08J3/24C07K1/14C07K17/08
Inventor 宓怀风邢小翠韩瑞芳
Owner NANKAI UNIV
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