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Method for simply, conveniently and quickly determining activity of alkaline phosphatase

A technology of phosphatase activity and assay method, applied in the field of alkaline phosphatase activity assay, can solve the problems of difficulty in realizing high-throughput, automation, and many steps, and achieve the effect of simple and rapid operation steps and high sensitivity

Inactive Publication Date: 2015-01-07
VAZYME BIOTECH NANJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This color-based method has many steps, requires precise control of reaction time, and is difficult to achieve high-throughput and automation

Method used

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  • Method for simply, conveniently and quickly determining activity of alkaline phosphatase
  • Method for simply, conveniently and quickly determining activity of alkaline phosphatase
  • Method for simply, conveniently and quickly determining activity of alkaline phosphatase

Examples

Experimental program
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Embodiment 1

[0019] Embodiment 1. Establishment of the method for measuring alkaline phosphatase activity by fluorescence method

[0020] 1. Preparation of Phosphorylated Double-Stranded DNA

[0021] Primer F (5' phosphorylation modification): 5'-aataacgtcggcaactttgg-3';

[0022] Primer R: 5'-gttacgccaccagtcatcct-3';

[0023] PCR template: λ DNA

[0024] PCR reaction:

[0025] components volume DDW to 50μl 10x Taq buffer 5 dNTPs (10 mM) 1μl Primer F (10 μM) 2μl Primer R (10 μM) 2μl lambda DNA 10ng Taq enzyme 1 U

[0026]

[0027] The PCR product was extracted with phenol-chloroform and purified by ethanol precipitation. The concentration of the purified PCR product was determined by A260.

[0028] 2. Alkaline phosphatase reaction:

[0029] components volume DDW to 20μl 10x CIAP buffer 2 Phosphorylated PCR product 100ng Bovine intestinal alkaline phosphatase (CIAP) 0-1024 mU

...

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Abstract

The invention discloses a method for simply, conveniently and quickly determining the activity of alkaline phosphatase. The method comprises the following steps: obtaining 5'-end phosphorylated double-stranded linear DNA (deoxyribonucleic acid) of one chain, then reacting in a reaction system in the presence of alkaline phosphatase by taking the double-stranded linear DNA as a substrate, reacting a reactant of the 5'-end phosphorylated double-stranded linear DNA obtained by reaction in the presence of sufficient gamma exonuclease, determining the relative amount of single-stranded DNA or double-stranded DNA in the reaction system after reaction, and deducing the degree of activity of alkaline phosphatase according to the detection result. Compared with the prior art, the method is simple and fast to operate, can realize high-throughput and automated activity determination, further has high sensitivity and can detect 10mU of alkaline phosphatase.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a simple and rapid alkaline phosphatase activity assay method. Background technique [0002] Alkaline phosphatase (alkaline phosphatase, AP) is a type of hydrolase, which can remove phosphate groups on molecules such as nucleotides, proteins, and alkaloids, and perform dephosphorylation. It is most effective in an alkaline environment, so it is named alkaline phosphatase. Alkaline phosphatase is a very important tool enzyme in genetic engineering technology and molecular biology research. It non-specifically catalyzes the dephosphorylation of 5' and 3' terminal phosphate monoesters of DNA and RNA, but does not degrade phosphodiester and triester bond. In addition, alkaline phosphatase can hydrolyze ribose and deoxyribonucleoside triphosphates (rNTPs and dNTPs). In molecular biology research, it can be used to remove phosphorylated ends of DNA and RNA for subse...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/42
CPCC12Q1/44C12Q1/42
Inventor 徐晓昱
Owner VAZYME BIOTECH NANJING
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