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Method for fixing egg white by utilizing polyelectrolyte

A polyelectrolyte brush and polyelectrolyte technology, which are applied in the directions of fixing on/in organic carriers, chemical instruments and methods, carrier binding/immobilization peptides, etc., can solve the problems of low utilization rate of interior space of brushes and the like

Active Publication Date: 2013-12-25
SHANGHAI MAG GENE NANOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another literature report uses polyacrylic acid brushes with a degree of polymerization of more than 1000 to bind RNase (RNase A) in the same way. Binding Kinetics and Activity, Langmuir, 2008, 24, 913-920), although the amount of binding has improved, but for such a high-length polyacrylic brush, the utilization rate of the internal space of the brush is still at a low level

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Chemical immobilization of bovine serum albumin (BSA protein) was performed using polyacrylic acid ball brushes.

[0039] First, the surface-initiated RAFT polymerization method published in "Journal of Colloid and Interface Science" in Volume 398, 2013, pages 82-87 was used to prepare silicon oxide with a spherical core of 100 nanometers, and the polyelectrolyte brush-like segment length was 40 nanometers. The branch density is 0.24nm -2 polyacrylic acid electrolyte ball brush.

[0040] By centrifugation-resuspension, the above-mentioned ball brush was washed twice with 10 mM MES buffer (pH=5.0, containing 0.05% w / v Tween-20), and ultrasonically dispersed in the above-mentioned buffer. At the same time, the buffer was used to dissolve the BSA protein to prepare a protein solution, which was added to the dispersion containing the ball brush and mixed evenly. The final ball brush concentration was 0.8 mg / ml, and the protein concentration was 1 mg / ml.

[0041] Incubate...

Embodiment 2

[0046] Lysozyme protein was chemically immobilized using polyacrylic acid ball brushes.

[0047] The polyelectrolyte brush used was the same as in Example 1.

[0048] By centrifugation-resuspension, the above-mentioned ball brush was washed twice with 10 mM MES buffer (pH=6.0, containing 0.05% w / v Tween-20), and ultrasonically dispersed in the above-mentioned buffer. At the same time, dissolve the lysozyme protein with the buffer solution to prepare a protein solution, add it into the dispersion liquid containing the ball brush, and mix evenly. The final ball brush concentration was 0.5 mg / ml and the protein concentration was 1.5 mg / ml.

[0049] Incubate at a constant temperature in an oven at 37°C for 2 hours, so that the protein is fully adsorbed on the surface of the polyelectrolyte brush.

[0050] Centrifuge, discard the supernatant, and wash the product 2 times with the above MES buffer. At the same time, the EDC was dissolved in the buffer solution to prepare an EDC s...

Embodiment 3

[0054] BSA proteins were chemically immobilized using poly-N-(2-aminoethyl)acrylamide ball brushes.

[0055] The synthesis method of the polyelectrolyte ball brush is the same as in Examples 1 and 2, wherein the monomer is replaced by N-(2-aminoethyl) acrylamide by acrylic acid to obtain a ball core of 100 nanometer silicon oxide, and the polyelectrolyte brush chain The segment length is 60 nm and the grafting density is 0.15 nm -2 polyacrylic acid electrolyte ball brush.

[0056] The chemical immobilization method of the ball brush is the same as that in Example 2, and the protein binding amount of the obtained poly N-(2-aminoethyl)acrylamide ball brush-BSA complex is 1500 μg BSA / mg ball brush as tested by the BCA method. It is 11 times of the monolayer saturation binding capacity, and the complex can exist stably under conditions such as PBS, and there is no protein release phenomenon.

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PUM

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Abstract

The invention discloses a method for fixing egg white by utilizing polyelectrolyte. According to the method, a polyelectrolyte brush with a 3D structure is adopted to serve as a carrier, the polyelectrolyte brush can adsorb a large amount of egg white under the condition benefiting the egg white physical adsorption of the polyelectrolyte brush, and then the egg white is stably bonded to the polyelectrolyte brush carrier through chemical bonds in a coupling chemistry mode. By means of the method, the combining quantity of the egg white is greatly improved, the purpose of stably combining the egg white through the polyelectrolyte brush is achieved, and the method can be effectively and efficiently applied to various downstream biotechnologies.

Description

technical field [0001] The invention relates to the technical field of biological materials, in particular to a protein immobilization method. Background technique [0002] The immobilization process of proteins plays an important role in many biotechnologies such as biosensors, protein separation and purification, diagnostic detection and analysis, and immobilized enzyme catalysis. In many applications, it is required that the carrier has a high protein binding capacity to improve the application performance of the carrier in the processes of separation, detection, analysis, and catalysis. The polyelectrolyte brush is a new type of carrier, which is composed of a carrier and a polyelectrolyte segment grafted on the surface of the carrier. Due to the extremely high density of the polyelectrolyte segment grafted on the surface of the carrier, in a good solvent, the polyelectrolyte chain The segments stretch out on the surface of the carrier to present a brush-like structure....

Claims

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Application Information

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IPC IPC(8): C07K17/08C12N11/08
Inventor 徐宏瞿振元古宏晨
Owner SHANGHAI MAG GENE NANOTECH CO LTD
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