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Centrifugal solid-phase enzyme cutting column, preparation method and application thereof

A spin column and solid-phase enzyme technology, applied in the biological field, can solve the problems of high enzyme cost, long use time, and large amount of protease, and achieve the effect of convenient separation, simple steps, and improved enzyme digestion efficiency

Inactive Publication Date: 2017-08-18
BEIJING MEIZHENG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 1. The current protease hydrolysis method takes too long, and the usual enzymatic hydrolysis time is about 20 hours to ensure sufficient enzymatic hydrolysis of the target protein
[0008] 2. The amount of protease used in the current method is relatively large, because the protease cannot be separated from the digested product, and the protease is a one-time use
The cost of the enzyme thus brought is high

Method used

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  • Centrifugal solid-phase enzyme cutting column, preparation method and application thereof
  • Centrifugal solid-phase enzyme cutting column, preparation method and application thereof
  • Centrifugal solid-phase enzyme cutting column, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0083] Example 1: Preparation of Centrifugal Solid-phase Digestion Column by Trypsin

Embodiment approach

[0084] A preferred embodiment of the preparation of the present invention utilizing trypsin to prepare a centrifugal solid-phase enzymatic digestion column is as follows:

[0085] 1. Vector activation

[0086] Select N-hydroxysuccinimide (NHS) modified agarose carrier sepharose4FF for activation

[0087] Take 1g of N-hydroxysuccinimide-modified agarose gel sepharose4FF, add 50ml of 1mM HCl, after swelling for 30min, wash the gel with 100ml of 1mM HCl for 6 times, and then wash with 100ml of ultrapure water

[0088] 2. The activated agarose gel sepharose4B with coupling buffer (0.1M NaHCO 3 , 0.8M NaCl, pH8.2) and washed 3 times. Add 20mol / L methylated trypsin, and couple at room temperature for 2 hours

[0089] 3. Wash the coupled trypsin-agarose carrier with 20mM, pH7.4 phosphate buffer PBS 3 times

[0090] 4. closed

[0091] Add 100mmol / L Tris.Cl pH8.0 to the coupled trypsin-agarose carrier, and react at room temperature for 2 hours

[0092] 5. Wash the blocked trypsin...

Embodiment 2

[0101] Example 2: Digestion of bovine lactoferrin by centrifugal trypsin solid-phase digestion column

[0102] 1. Take 10mg of bovine lactoferrin and prepare 10mg / ml lactoferrin solution with 100mM NH4HCO3, PH 8.0 buffer

[0103] 2. Take a centrifugal trypsin solid-phase enzymatic digestion column described in the present invention, cover the upper and lower plugs

[0104] 3. Take 0.1ml of the above-prepared bovine lactoferrin solution, add it to the above solution, and add 0.1ml of 6M Guanidine and 20ul of 1M DTT. Shake to mix for 5 seconds

[0105] 4. The above-mentioned spin column was reacted at 37°C for 30 minutes at a constant temperature.

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Abstract

The invention provides a centrifugal solid-phase enzyme cutting column, a preparation method and an application thereof. Protease fixed on carriers is loaded into a centrifugal column component. The centrifugal column component comprises a column pipe, a sieving plate, a sealing ring, an upper cover, a lower plug and a column pipe sleeve. The protein to be enzyme-cut can finish enzyme cutting reaction in the centrifugal column. After the enzyme cutting reaction is finished, products after enzyme cutting are separated by utilizing a centrifugal or positive-negative pressure separation mode. The enzyme cutting efficiency is high, and no pollution of enzyme molecules is caused in the products. Compared with the traditional enzyme cutting method, the enzyme cutting efficiency can be improved, and the enzyme can be repeatedly utilized, so that the enzyme cutting cost is greatly reduced.

Description

technical field [0001] The invention provides a preparation method and application of a centrifugal solid-phase enzyme digestion column. The invention belongs to the field of biotechnology, in particular to the enzymatic hydrolysis and analysis of protein molecules. Background technique [0002] With the rise of proteomics research, the fine analysis of proteins has attracted more and more interest of researchers. Proteomics is the study of the composition, activity and protein-protein interaction of intracellular proteins from an overall level, and is a new discipline in the era of functional genomics. At present, there are two main routes for proteomics research: one is proteomics based on two-dimensional electrophoresis; the other is proteomics based on mass spectrometry, and the proteomics research route based on two-dimensional electrophoresis is ultimately inseparable from the application of mass spectrometry technology. . [0003] Mass spectrometry analysis include...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/40C12P21/06
CPCC12M21/18C12P21/06
Inventor 张彦明
Owner BEIJING MEIZHENG BIOLOGICAL TECH
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