Method of detecting protein palmitoylation modification locus

A detection method and palmitoylation technology, applied in the field of protein post-translational modification detection, can solve the problems of difficult to purify protein samples, limited analysis samples, complicated operations, etc. stable effect

Inactive Publication Date: 2019-03-29
XINXIANG MEDICAL UNIV
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  • Abstract
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Problems solved by technology

The wide application of mass spectrometry to directly detect the palmitoylation modification sites of proteins, as well as the development of acyl-biotinyl exchange (ABE) method and metabolic labeling method of palmitate analogues, make more and more The site of palmitoylation modification has been identified, but the existing methods also have their own shortcomings: it is not easy to obtain purified protein samples, or the sample purification process is easily lost, so that the palmitoylation modification cannot be detected, or the palmitoylation modification The protein is unstable, or the operation is cumbersome, the sensitivity is not high, and the analysis samples are limited, etc.

Method used

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  • Method of detecting protein palmitoylation modification locus
  • Method of detecting protein palmitoylation modification locus
  • Method of detecting protein palmitoylation modification locus

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Embodiment 1

[0046] 1Purification of target protein

[0047] 1.1 Sample preparation

[0048] The APT1 protein with Flag tag was overexpressed in HEK-293T cells, and the Flag tag encoded a hydrophilic polypeptide DYKDDDDK of 8 amino acids. The protein was extracted with Beyond P0013IP cell lysate to obtain APT1 protein solution, the measured concentration was 3mg / ml, and 30mg of the protein solution was taken.

[0049] 1.2 Purified protein

[0050] Mix the anti-DYKDDDDK tag affinity medium (Sino Biological, Catalog: 101274-MM13-RN) upside down, take 150μl medium and add 15ml containing 3ml equilibration buffer (10mMPBS, pH 7.4) In a centrifuge tube, centrifuge at 1000g for 3 minutes (washing medium); repeat washing twice with 3ml of equilibration buffer (avoid taking out the medium); add about 10ml of target protein solution to the washed medium, and place it on a rotator at 4°C overnight; The protein solution was centrifuged at 1000g for 3min, and the supernatant was removed; the precip...

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Abstract

The invention relates to a method of detecting a protein palmitoylation modification locus. After target protein is purified, an LC-MS/MS technology is used for palmitoylation modification locus identifying on the target protein. The method is simple and easy to operate and strong in maneuverability, and the problems that protein palmitoylation modification is instable and a sample is easy to getlost can be solved.

Description

technical field [0001] The invention relates to the detection field of protein post-translational modification, in particular to a high-throughput, high-reliability detection method for protein palmitoylation modification sites. Background technique [0002] Protein is the basic functional unit to perform cell functions, and also the direct executor of life activities. Proteins need to undergo different degrees of translational modification to exert corresponding physiological functions. After translation modification, the protein structure is more complex and the function is more perfect. This process widely exists in life activities, and at the same time, it is important for the normal operation of a series of life activities such as material transportation, genetic material replication, cell proliferation and differentiation, and cell apoptosis. have an important safeguarding role. When the function of the protein is abnormal, its life activities will be disordered and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6803
Inventor 张中健孔二艳
Owner XINXIANG MEDICAL UNIV
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