Transhydrogenase-1 activity determination kit and use method thereof

An activity measurement and transhydrogenase technology, applied in the field of life sciences, can solve the problem of not detecting transhydrogenase-1, etc., and achieve the effect of eliminating the interference of NAD, high detection specificity and good repeatability

Inactive Publication Date: 2018-02-23
SUZHOU COMIN BIOTECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

At present, there is no effective method for detecting transhydrogenase-1 (TH-1) activity on the market

Method used

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Embodiment Construction

[0077] The present invention will be described in detail below in conjunction with embodiments.

[0078] A transhydrogenase-1 activity assay kit, comprising the following reagents:

[0079] Reagent 1, liquid 100mL×1 bottle, made by mixing Tris-HCl buffer solution, sucrose and EDTA, placed in a 100mL volumetric flask, stored at -20°C;

[0080] Reagent 2, liquid 50mL×1 bottle, composed of Tris-HCl buffer solution, placed in a 50mL volumetric flask, stored at -20°C;

[0081] Reagent 3, liquid 18mL×1 bottle, composed of Tris-HCl buffer solution, placed in a 20mL reagent bottle, stored at 4°C;

[0082] Reagent 4, powder × 1 tube, composed of NADH, placed in a 1.5mL EP tube, stored at -20°C;

[0083] Reagent 5, powder × 1 tube, composed of AcPyADP, placed in a 1.5mL EP tube, stored at -20°C.

[0084] Further, the substance concentration of the Tris-HCl buffer solution contained in the reagent one is 10 mM, the pH is 7.5, the volume is 100 mL, the mass of sucrose is 8.56 mg, and t...

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Abstract

The invention discloses a transhydrogenase-1 activity determination kit and a use method thereof. The kit comprises a mixed solution of a Tris-HCl buffer solution, sucrose and EDTA, a Tris-HCl buffersolution, NADH and AcPyADP. The method is based on the detection principle of replacing the NADP<+> with a synthetic substrate 3-acetylpyridine adenine dinucleotide phosphate (APADP<+>) and calculating TH-1 activity through measuring a light absorption increasing speed at 375nm of APADPH generated by the reduction of APADP<+> catalyzed by TH-1, can effectively eliminate the interference of NAD andhas high detection specificity. Through a simple work liquid, only through adding a sample and a work liquid into a microcuvette or a 96-well plate, the determination is finished and detection is convenient. The kit can effectively isolate cytoplasmic proteins and mitochondrial proteins and accurately determine TH-1 located in the mitochondrial inner membrane.

Description

technical field [0001] The invention belongs to the field of life sciences, and in particular relates to a transhydrogenase-1 activity assay kit and a method thereof. Background technique [0002] Transhydrogenase (TH) is located on the inner membrane of mitochondria, also known as respiratory electron transport chain complex six, which catalyzes NADH+NADP + and NAD + +NADPH is interconverted. Catalyzing the forward reaction is called transhydrogenase-1 (TH-1). Increased mitochondrial NADH content leads to H in the mitochondrial membrane + The electrochemical gradient is elevated, thereby promoting the generation of ROS along the electron transport chain. Transhydrogenase-1 (TH-1) promotes the conversion of NADH to NADPH, thereby improving the antioxidant capacity of mitochondria. [0003] Both NADH and NADPH have characteristic light absorption at a wavelength of 340nm, so the hydrogenation reaction catalyzed by transhydrogenase (TH) cannot cause a change in the absorb...

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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