Preparation method of biotin labeled inorganic pyrophosphatase

An inorganic pyrophosphatase and biotin labeling technology, applied in the biological field, can solve the problem of low activity of biotinylated proteins, and achieve the effect of high throughput and long reading.

Inactive Publication Date: 2015-11-18
BEIJING ZHONGKEZIXIN TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, biotinylation methods include chemical modification and biological mod

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] (1) Use Tris-HCl buffer solution with pH 8.5 to dissolve the inorganic pyrophosphatase, the concentration of the inorganic pyrophosphatase is 1.8mg / ml;

[0026] (2) Activation of biotin: Add 1 mol of biotin, 1.25 mol of EDC, and 1 mol of NHS into 1L of analytically pure dimethylformamide to activate biotin;

[0027] (3) Equilibrate biotin to 10°C;

[0028] (4) Dissolve 0.24mol of activated biotin in a mixed solvent obtained by mixing 90ml of dimethylformamide and 100ml of tetrahydrofuran, add 0.01mol of inorganic pyrophosphatase, mix and stir for 8 hours at 4°C to obtain a mixed solution;

[0029] (5) Use 1LTris-HCl buffer to dialyze 100ml of the mixed solution for 18 hours at a temperature of 4°C to remove impurities and free biotin, and replace the Tris-HCl buffer every 6 hours;

[0030] (6) Determination of protein content using absorbance at 280nm;

[0031] (7) Add a protective agent twice the volume of the enzyme. The protective agent contains 10mMTris-HCl buffe...

Embodiment 2

[0033] (1) Use Tris-HCl buffer solution with pH 8.5 to dissolve the inorganic pyrophosphatase, the concentration of the inorganic pyrophosphatase is 1.5mg / ml;

[0034] (2) Activation of biotin: Add 1 mol of biotin, 1.25 mol of EDC, and 1 mol of NHS into 1L of analytically pure dimethylformamide to activate biotin;

[0035] (3) Equilibrate biotin to 4°C;

[0036] (4) Dissolve 0.22mol of activated biotin in a mixed solvent obtained by mixing 90ml of dimethylformamide and 100ml of tetrahydrofuran, add 0.01mol of inorganic pyrophosphatase, mix and stir for 5 hours at 4°C to obtain a mixed solution;

[0037] (5) Use 1LTris-HCl buffer to dialyze 80ml of the mixed solution for 12 hours at a temperature of 4°C to remove impurities and free biotin, and replace the Tris-HCl buffer every 4 hours;

[0038] (6) Determination of protein content using absorbance at 280nm;

[0039] (7) Add a protective agent 1.5 times the volume of the enzyme, the protective agent contains 10mM Tris-HCl bu...

Embodiment 3

[0041] (1) Use Tris-HCl buffer solution with pH 8.5 to dissolve the inorganic pyrophosphatase, the concentration of the inorganic pyrophosphatase is 1.6mg / ml;

[0042] (2) Activation of biotin: add 1mol of biotin, 1.25mol of EDC, and 1mol of NHS to analytically pure dimethylformamide to activate biotin;

[0043] (3) Equilibrate biotin to 4°C;

[0044] (4) Dissolve 0.25 mol of activated biotin in a mixed solvent of dimethylformamide and tetrahydrofuran, add 0.01 mol of inorganic pyrophosphatase, mix and stir for 6 hours at 4°C to obtain a mixed solution;

[0045] (5) Dialyze 90ml of the mixed solution with 1LTris-HCl buffer solution for 15 hours at 4°C to remove impurities and free biotin, and replace the Tris-HCl buffer solution at 4 hours and 8 hours;

[0046] (6) Determination of protein content using absorbance at 280nm;

[0047] (7) Add a protective agent twice the volume of the enzyme, which contains 10mM Tris-HCl buffer, 20% glycine, 0.2mM mannitol, 0.15mMDTT and 0.08m...

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Abstract

The invention belongs to the technical field of biotechnology and particularly relates to a preparation method of biotin labeled inorganic pyrophosphatase. The method includes the steps of dissolving inorganic pyrophosphatase, activating biotin, combining the biotin with the inorganic pyrophosphatase, performing dialyzing, measuring a protein content, and storing. The inorganic pyrophosphatase prepared by the method is very suitable for application in pyrosequencing reactions; high activity of the inorganic pyrophosphatase ensures accurateness of the pyrosequencing reactions.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of biotin-labeled inorganic pyrophosphatase. Background technique [0002] Biotinylation is the process of covalently attaching biotin to proteins, nucleic acids, or other molecules. Due to the small molecular weight of biotin (molecular weight is 244.31), the biotinylation reaction is fast, efficient and not easily disturbed. Biotinylated molecules can interact with streptavidin and avidin through biotin, and are not affected by high heat, pH, and proteolysis. The binding of biotin to streptavidin and avidin is specific and efficient, so this interaction has a wide range of applications in many fields of biotechnology. In addition, multiple biotinylated molecules can be cross-linked to form a protein of interest to a researcher, while allowing binding to multiple streptavidin, avidin, and neutravidin proteins. detection sensitivity. In addition, ...

Claims

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Application Information

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IPC IPC(8): C12N9/14
CPCC12N9/14C12Y306/01001
Inventor 高静蔡亦梅徐潇吴超张睿王者馥王绪敏殷金龙任鲁风
Owner BEIJING ZHONGKEZIXIN TECH
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