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Isocitrate lyase activity testing kit and its method

A technology for isocitrate and activity determination, applied in the field of life sciences, can solve problems such as false positives and large interference, and achieve the effects of low cost, good repeatability and improved detection efficiency

Inactive Publication Date: 2018-03-13
SUZHOU COMIN BIOTECH
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because pyruvate can also react with dinitrophenylhydrazine, the method is greatly disturbed and there are false positives. In scientific research, it is urgent to develop a new method to detect the activity of isocitrate lyase

Method used

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Embodiment Construction

[0056] The present invention will be described in detail below in conjunction with embodiments.

[0057] An isocitrate lyase activity assay kit, comprising the following reagents:

[0058] Extract solution, liquid 100mL×1 bottle, composed of Tris-HCl buffer solution, KCl, MnCl 2 , DTT and EDTA, and adjust the pH to 7.0, put in a 100mL volumetric flask, and store at 4°C;

[0059] Reagent 1, liquid 15mL×1 bottle, made by mixing disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution and water, placed in a 15mL reagent bottle, stored at 4°C;

[0060] Reagent 2, powder x 1 bottle, made of MgCl 2 ·6H 2 O, DTT and NADH are mixed, placed in a 1.5mL EP tube, and stored at -20°C;

[0061] Reagent 3, liquid 360μL x 1 tube, composed of lactate dehydrogenase, placed in a 1.5mL EP tube, stored at 4°C;

[0062] Reagent 4, powder x 1 bottle, composed of isocitric acid, placed in a 5mL reagent bottle, stored at 4°C.

[0063] Further, the substance concentration of the Tr...

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Abstract

The invention discloses an isocitrate lyase activity testing kit and its method. The kit comprises solution prepared by mixing Tris-HCl buffer solution, KCl, MnCl2, DTT and EDTA, solution prepared bymixing disodium hydrogen phosphate-monosodium phosphate buffer solution and water, powder formed by mixing MgCl2.6H2O, DTT and NADH, lactic dehydrogenase and isocitric acid. The testing principle of the method includes steps of degrading ICL catalytic isocitrate to be glyoxylic acid and succinic acid; making glyoxylic acid and NADH generate ethanol and NAD under the LDH effect, wherein the NADH has feature absorption peak under 340 nm, and the reducing rate of the 340 nm light absorbance can indirectly reflect the ICL activity. Through the enzyme coupling effect, the ICL activity can be indirectly reflected through detecting the NADH content under the ultraviolet wave length, the disturbance of colorful matter is effectively removed, and the detection accuracy is greatly improved.

Description

technical field [0001] The invention belongs to the field of life sciences, and in particular relates to an isocitrate lyase activity assay kit and a method thereof. Background technique [0002] Isocitrate lyase (ICL) mainly exists in plants and microorganisms. During the germination process of oil crop seeds, fat is converted into carbohydrates through the glyoxylate cycle and other processes, and isocitrate lyase is the glyoxylate cycle. one of the key enzymes. [0003] Isocitrate lyase catalyzes the degradation of isocitrate into glyoxylate and succinate, and the change of glyoxylate can indirectly reflect the activity of isocitrate lyase. At present, the most widely used method for measuring the activity of isocitrate lyase is the colorimetric method, that is, glyoxylic acid and dinitrophenylhydrazine react to form glyoxylic acid phenylhydrazine, which is brown under alkaline conditions, and the color depth is similar to that of glyoxylic acid. The content is proporti...

Claims

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Application Information

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IPC IPC(8): G01N21/31
CPCG01N21/31
Inventor 姚金美赵林川
Owner SUZHOU COMIN BIOTECH
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