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Non-diagnosis-purpose method for detecting ApoA5 biological activity and kit

A biologically active and active technology, applied in biochemical equipment and methods, microbial determination/inspection, measurement devices, etc., can solve the problem of low detection of ApoA5

Inactive Publication Date: 2015-04-01
THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are few methods for detecting the biological activity of ApoA5

Method used

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  • Non-diagnosis-purpose method for detecting ApoA5 biological activity and kit
  • Non-diagnosis-purpose method for detecting ApoA5 biological activity and kit
  • Non-diagnosis-purpose method for detecting ApoA5 biological activity and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Detection of ApoA5 Biological Activity

[0033] 125 Preparation of I-ApoA5: using the Iodogen method, using Na 4 I to label ApoA5, ​​use Sophohdex G200 column to elute free 125 I, prepared with 125 I-labeled ApoA5 protein.

[0034] HL-1 cardiomyocytes were seeded in a 12-well plate at 1×10 6 / cm 2 After the cells reached 90% confluence, the medium was changed and starved for 12 hours, and the following experimental groups were performed: ① control group: adding claycomb medium containing 1% serum; ② LRP1 silencing group: adding 5nmol / L LRP1 small interfering RNA 1% serum claycomb medium to silence the expression of LRP1 on the HL-1 cell membrane. After 6 hours the medium was aspirated and complete claycomb medium was added.

[0035] After 48 hours, add 125 I-ApoA5 (about 2×l0 6 cpm / well), and incubated together for 2 hours. Aspirate the medium and wash 3 times with pre-cooled PBS. Then add heparin solution (10 mg / ml) and incubate at room temperature...

Embodiment 2

[0042] Example 2 Detection of ApoA5 Biological Activity

[0043] Preparation of adipocytes: Inoculate mouse-derived 3T3-L1 preadipocytes in vitro into 6-well plates. When the cells are 90% full, add 0.5mmol / L IBMX+10μg / mL insulin + 0.25 μmol / L dexamethasone, incubated for 9 days, induced to differentiate into mature adipocytes.

[0044] 125 Preparation of I-ApoA5: using the Iodogen method, using Na 4 I to label ApoA5, ​​use Sophohdex G200 column to elute free 125 I, prepared with 125 I-labeled ApoA5 protein.

[0045] The differentiated and mature adipocytes were starved for 12 hours, and the following experimental groups were carried out: ① control group: adding serum-free DMEM medium; ② LRP1 silencing group: adding serum-free DMEM medium containing 5 nmol / L LRP1 small interfering RNA, To silence the expression of LRP1 on the adipocyte membrane. After 6 hours, the medium was aspirated, and complete DMEM medium was added.

[0046] After 48 hours, add 125 I-ApoA5 (about...

Embodiment 3

[0052] Example 3 Detection of ApoA5 Biological Activity

[0053] 125 Preparation of I-ApoA5: using the Iodogen method, using Na 4 I to label ApoA5, ​​use Sophohdex G200 column to elute free 125 I, prepared with 125 I-labeled ApoA5 protein.

[0054] HL-1 cardiomyocytes were seeded in a 12-well plate at 1×10 6 / cm 2 After the cells reached 90% confluence, the medium was changed and starved for 12 hours, and the following experimental groups were performed: ① control group: add claycomb medium containing 1% serum; ② LRP1 silencing group: add small interfering RNA containing 10nmol / L LRP1 1% serum claycomb medium to silence the expression of LRP1 on the HL-1 cell membrane. After 6 hours the medium was aspirated and complete claycomb medium was added.

[0055] After 48 hours, add 125 I-ApoA5 (about 2×l0 6 cpm / well) and incubated for 1 hour. Aspirate the culture medium and wash 3 times with pre-cooled PBS. Then add heparin solution (10mg / ml) and incubate at room temperatu...

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Abstract

The invention relates to the field of protein activity detection, in particular to a non-diagnosis-purpose method for detecting ApoA5 biological activity and a kit. The method comprises the steps that a cell and an ApoA5 marked through radionuclide are incubated, the ApoA5 combined on the cytomembrane is removed, the cell is split, the radioactive strength and the cell protein concentration of the cell lysis solution are detected, and the first radioactive activity is obtained; the cell and the LRP1 small interfering RNA are mixed, and the cell expressed by the silence LRP1 is obtained; the cell expressed by the silence LRP1 and the ApoA5 marked through the radionuclide are incubated, the ApoA5 combined on the cytomembrane is removed, the cell is split, the radioactive strength and the cell protein concentration of the cell lysis solution are detected, and the second radioactive activity is obtained; the first radioactive activity and the second radioactive activity are compared. According to the detection method, the biological activity of the ApoA5 can be accurately detected by observing the uptake situation of the HL-1 myocardial cell to the ApoA5.

Description

technical field [0001] The invention relates to the field of protein activity detection, in particular to a method and kit for non-diagnostic detection of ApoA5 biological activity. Background technique [0002] With the improvement of modern living standards and changes in lifestyles, the incidence of obesity is increasing. At the same time, the increase in lipids and ectopic deposition associated with obesity makes the incidence of obesity-related diseases increase year by year. Therefore, effectively reducing the lipid deposition associated with obesity is an important challenge facing medicine today. [0003] Obesity is the result of excessive storage of triglyceride (TG) in adipocytes due to the imbalance between energy intake and energy consumption, and the result of abnormal increase in cell volume. The root cause is abnormal lipid metabolism. The essence of abnormal lipid metabolism refers to the abnormality of lipoprotein and apolipoprotein. The activity of key en...

Claims

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Application Information

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IPC IPC(8): C12Q1/02G01N23/00
Inventor 赵水平罗俊郑小燕聂赛赵旺
Owner THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV