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Dot blotting method for detecting activity of GSK-3beta proteins of human blood platelets

A platelet and β protein technology, applied in the field of biomedicine, can solve the problem of lack of ideal monitoring methods for GSK-3β protein activity, and achieve the effect of simple operation

Inactive Publication Date: 2015-06-10
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no ideal monitoring method for the activity of GSK-3β protein in human peripheral blood

Method used

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  • Dot blotting method for detecting activity of GSK-3beta proteins of human blood platelets
  • Dot blotting method for detecting activity of GSK-3beta proteins of human blood platelets
  • Dot blotting method for detecting activity of GSK-3beta proteins of human blood platelets

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Experimental program
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Embodiment 1

[0031] Embodiment 1 Establishment of the Dot-blot method of the present invention

[0032] 1. Platelet separation and purification.

[0033] 1.1 Human whole blood 5ml, 60g, centrifuged at 4 degrees Celsius for 15 minutes, the upper layer is plasma, the middle layer is white blood cells, and the lower layer is red blood cells. Plasma, 60g, centrifuged at 4°C for 15min, to remove sediment, centrifuged at 1500g for 15min, divided into 2 layers, the upper layer was serum, and the lower layer was platelets.

[0034] 1.2 Platelet samples were washed with modified Tyrode's solution, centrifuged at 1500g for 5min three times, and stored in a -80°C refrigerator.

[0035] 2. Platelet protein extraction and concentration determination.

[0036] 2.1 Add 120 μl of pre-cooled lysate to platelet 2×Buffer, the 2×Buffer is composed of 50mM Tris-Cl pH 8.0, 1% NP-40, 150mM NaCl, 0.1% SDS, 0.02% NaNR3R, 20% glycerol, cocktail, Composed of 1% PMSF, after standing on ice for 20 minutes, the prot...

Embodiment 2

[0046] Comparison of platelet GSK-3β activity in type 2 diabetes mellitus (T2DM) group and patients with T2DM and MCI detected by dot blot

[0047] 1. Experimental methods: ① Inclusion criteria: Diabetic patients aged >50 years old; no visual and hearing impairments that significantly affect cognitive tests; no history of severe hypoglycemia, diabetic ketosis coma, and hyperosmolar coma; no alcohol dependence and psychoactivity History of substance abuse; no brain trauma, no cardiovascular and cerebrovascular events, including cerebral infarction, cerebral hemorrhage, TIA, myocardial infarction, etc.; no other serious physical diseases that affect cognitive tests, such as epilepsy, hypothyroidism, hypoxemia symptoms, etc.; no history of mental illness; voluntary participation in this study, able to cooperate with the examination, and good compliance. ②Study groups: type 2 diabetes without cognitive impairment T2DM group (T2DM), type 2 diabetes with mild cognitive impairment MC...

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Abstract

The invention provides a dot blotting method for detecting the activity of GSK-3beta proteins of human blood platelets. The dot blotting method comprises the steps of preparing the proteins of the blood platelets to realize the uniform concentration, drying sample dots on an NC film in air, closing by 5 percent of skim milk powder, then respectively adding primary antibodies for 1: 1000GSK-3beta and Ser9-GSK-3beta, performing dilution, adding 1:10000 fluorescent secondary antibodies marked with IRDyeT 800 channels of the Licor Company, performing dilution again, and finally scanning with fluorescence. According to the antigen and antibody combination reaction theory, the method disclosed by the invention can be used for performing simple and quick quantitative analysis on the activity of the GSK-3beta proteins and can be used for diagnosing and preventing senile dementia.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a dot blot method capable of detecting the activity of human platelet GSK-3β protein. Background technique [0002] Glycogen synthase kinase 3 (Glycogen Synthase Kinase 3, GSK-3) is a highly conserved serine / threonine protein kinase, ubiquitously present in mammalian eukaryotic cells. GSK-3 is an important regulator of insulin-dependent glycogen synthesis, and it can also act on many signaling protein structural proteins and transcription factors to regulate cell differentiation, proliferation, survival and apoptosis. [0003] There are two main subtypes of GSK-3, namely GSK-3α and GSK-3β, with molecular weights of 51kDa and 47kDa, respectively. The two have 98% homology in the catalytic domain, and have slight differences in the N- and C-termini. Although both isoforms of GSK-3 are involved in neuropsychiatric disorders, most studies have focused on its beta isof...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6893G01N2800/042G01N2800/2814
Inventor 王建枝徐志鹏
Owner HUAZHONG UNIV OF SCI & TECH
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