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Preparation method for biotinylation luciferase

A technology of luciferase and biotinylation, which is applied in the field of preparation of biotinylated luciferase, can solve the problem of low biotinylated protein activity, and achieve the effects of ensuring accuracy, large read size, and high throughput

Inactive Publication Date: 2015-09-23
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 modification, but the activity of modified biotinylated proteins is generally not high

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] (1) Dissolve luciferase in PBS buffer with pH 7.4, the luciferase concentration is 1.8mg / ml;

[0023] (2) Activation of biotin: add 1 mol of biotin, 1.5 mol of DCC, 1.5 mol of NHS into 1L of analytically pure dimethylformamide to activate biotin;

[0024] (3) Dissolve 0.24mol of activated biotin in a mixed solvent obtained by mixing 100ml of dimethylformamide and 80ml of tetrahydrofuran, add 0.01mol of luciferase, mix and stir for 8 hours at 4°C to obtain a mixed solution ;

[0025] (4) Use 1L of PBS buffer to dialyze 100ml of the mixed solution for 18 hours at 4°C to remove impurities and free biotin, and replace the PBS buffer every 6 hours;

[0026] (5) Determination of protein content using absorbance at 280nm;

[0027] (6) Add a protective agent twice the volume of the enzyme, which contains 10mM PBS buffer, 20% glycine, 30% histidine, 0.15mM DTT, 0.08mM EDTA and 0.06mM sodium azide, and store in cold storage.

Embodiment 2

[0029] (1) Use pH 7.4 PBS buffer to dissolve luciferase, the concentration of luciferase is 1.5mg / ml;

[0030] (2) Activate biotin: add 1mol biotin, 1.5mol DCC, 1.5mol NHS to analytically pure dimethylformamide to activate biotin;

[0031] (3) Dissolve 0.22 mol of activated biotin in a mixed solvent of dimethylformamide and tetrahydrofuran, add 0.01 mol of luciferase, mix and stir for 5 hours at 4°C to obtain a mixed solution;

[0032] (4) Dialyze 80ml of the mixed solution with 1L of PBS buffer for 12 hours at 4°C to remove impurities and free biotin, and replace the PBS buffer every 4 hours;

[0033] (5) Determination of protein content using absorbance at 280nm;

[0034] (6) Add a protective agent 1.5 times the volume of the enzyme, the protective agent contains 10mM PBS buffer and 0.1mM DTT, and store in cold storage.

Embodiment 3

[0036] (1) Use pH 7.4 PBS buffer to dissolve luciferase, the luciferase concentration is 1.6mg / ml;

[0037] (2) Activate biotin: add 1mol biotin, 1.5mol DCC, 1.5mol NHS to analytically pure dimethylformamide to activate biotin;

[0038] (3) Dissolve 0.25 mol of activated biotin in a mixed solvent of dimethylformamide and tetrahydrofuran, add 0.01 mol of luciferase, mix and stir for 6 hours at 4°C to obtain a mixed solution;

[0039] (4) Dialyze 90ml of the mixed solution with 1L of PBS buffer for 15 hours at 4°C to remove impurities and free biotin, and replace the PBS buffer at 4 and 8 hours;

[0040] (5) Determination of protein content using absorbance at 280nm;

[0041] (6) Add a protective agent twice the volume of the enzyme, which contains 10mM PBS buffer, 20% glycine, 0.2mM mannitol, 0.15mM DTT and 0.08mM EDTA, and store in cold storage.

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method for biotinylation luciferase. The method comprises the following steps that 1, luciferase is dissolved by using a PBS buffer solution; 2, biotin is activated; 3, the activated biotin is dissolved in mixed solvent, the luciferase is added, and stirring is performed; 4, dialysis is performed on a mixed solution by using the PBS buffer solution; 5, the protein content is measured by using the 280 nm absorbancy; 6, a protective agent is added, and cold preservation is performed. According to the preparation method for the biotinylation luciferase, the obtained biotinylation luciferase is high in biological activity.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a preparation method of biotinylated luciferase. 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, there are a large number of ...

Claims

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Application Information

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IPC IPC(8): C12N9/02
CPCC12N9/0069C12Y113/12007
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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