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Method for detecting activity of methylenetetrahydrofolate dehydrogenase 2

A technology of methylenetetrahydrofolate and dehydrogenase, which is applied in the measurement of color/spectral characteristics, etc., can solve the problems of long reaction time and achieve the effect of shortening the detection time

Pending Publication Date: 2022-06-03
深圳市乐土生物医药有限公司
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Problems solved by technology

Although this method can effectively detect MTHFD2 activity; however, the reaction time is long, requiring at least 90 minutes; and the reaction conditions are limited by the properties of chemiluminescent reagents, so it is necessary to purchase GLO Detection system detection reagents, etc.

Method used

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  • Method for detecting activity of methylenetetrahydrofolate dehydrogenase 2
  • Method for detecting activity of methylenetetrahydrofolate dehydrogenase 2
  • Method for detecting activity of methylenetetrahydrofolate dehydrogenase 2

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Embodiment

[0033] 1. Main reagents and instruments

[0034] The main reagents and instruments used in this example are shown in Table 1.

[0035] Table 1 Main reagents and instruments

[0036] product name Specifications manufacturer Factory code Recombinant Human MTHFD2 Protein 0.5mg novusbio NBP2-51979-0.5mg LY 345899 5mg medchemexpress HY-101943 NAD+ 500mg medchemexpress HY-B0445 Folitixorin 50mg Shanghai Yifei Biological E044315 96-well transparent culture plate NEST 701001 Microplate reader Thermo VARIOSKAN LUX NADH 1g Germany 1.01E+10 HEPES 500g Aladdin H109406-500g

[0037] The buffers used in this example include:

[0038] Buffer stock solution:

[0039] 10mmol / L NADH: the molecular weight is 709.4, weigh 0.007094g, dissolve it to 1mL with sterile water, dispense 100μL / tube, and store in a -20 degree refrigerator.

[0040] 20mmol / L NAD+: molecular weight 663, weigh 0.01326g, di...

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Abstract

The invention discloses a method for detecting the activity of methylenetetrahydrofolate dehydrogenase 2. According to the activity detection method, methylenetetrahydrofolate dehydrogenase 2 to be detected and a catalyst NAD + cofactor are adopted to react with a reaction substrate 5, 10-methylenetetrahydrofolate, in the reaction process, 340 nM ultraviolet light absorption value detection is conducted on a reaction product, and the content of NADH in the reaction product is calculated according to the 340 nM ultraviolet light absorption value. And calculating the activity of the methylenetetrahydrofolate dehydrogenase 2 according to the NADH content after different reaction times. According to the method disclosed by the invention, the content of NADH in the reaction product can be obtained only by detecting the 340nM ultraviolet light absorption value in the reaction product according to an NADH standard curve, so that the activity of the methylenetetrahydrofolate dehydrogenase 2 is calculated. According to the method, the detection time is greatly shortened, and the method can be better suitable for scenes where the activity of the methylenetetrahydrofolate dehydrogenase 2 needs to be rapidly detected.

Description

technical field [0001] The present application relates to the technical field of polypeptide activity detection, in particular to a method for detecting the activity of methylenetetrahydrofolate dehydrogenase 2. Background technique [0002] Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), also known as methylenetetrahydrofolate cyclohydrolase, is a dual function with the dual activities of methylenetetrahydrofolate dehydrogenase and cyclohydrolase An enzyme that is highly expressed in mitochondrial folate metabolism. Studies have shown that the activity of MTHFD2 is closely related to the occurrence and development of various types of tumors; therefore, the detection of MTHFD2 activity is of great significance for tumor research. [0003] At present, the commonly used MTHFD2 activity detection method is to use the catalyst NAD+ cofactor and MTHFD2 to catalyze the dehydrogenation of 5,10-methylenetetrahydrofolate. After 30 minutes of reaction at room temperature, NADH is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/33
CPCG01N21/33
Inventor 宋侠侠何裕华朱锡帧姜孝明
Owner 深圳市乐土生物医药有限公司
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