A method for identifying protein phosphorylation sites

A phosphorylation and protein technology, which is applied in the field of identifying protein phosphorylation sites, can solve problems such as heavy workload, and achieve the effect of speeding up the research process and saving preparation costs

Active Publication Date: 2022-03-01
BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The standard approach for phosphorylation site analysis is to use 32 P marks the purified phosphorylated protein, separates the obtained peptides by one-dimensional thin-layer electrophoresis and two-dimensional thin-layer chromatography (called two-dimensional peptide spectrum), and detects phosphorylated peptides by autoradiography. Edman degradation sequencing; or use fluorescently labeled phosphopeptides. Then identify amino acids released from phosphorylated sites by specific retention times of phosphorylated residues, release of fluorescently labeled or radioactively labeled amino acids, and this method performs large-scale When applied at scale, the workload is obviously too large

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  • A method for identifying protein phosphorylation sites
  • A method for identifying protein phosphorylation sites
  • A method for identifying protein phosphorylation sites

Examples

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Embodiment 1

[0050] This embodiment provides a method for identifying protein phosphorylation sites, which includes the following steps.

[0051] (1) Determine the phosphorylation site of the sample to be tested

[0052] Most of the possible reversible phosphorylation sites that may exist are screened through literature search or phosphoproteomics analysis.

[0053] (2) Construction of expression vectors 1 to 3

[0054] The expression vectors 1 to 3 are plasmids respectively containing amino acid sequence fragments 1 to 3, and the amino acid sequence fragments 1 to 3 are all sequence fragments containing the detection site of the protein to be detected, and the sequence lengths are the same.

[0055] Among them, the amino acid sequence fragment 1 is a fragment in which most of the possible reversible phosphorylation sites except the site to be tested are subjected to permanent non-phosphorylation site mutations on the fragment of the protein to be tested;

[0056] Amino acid sequence fra...

Embodiment 2

[0061] This embodiment provides a method for detecting phosphorylation at a single point of ATGL, and this embodiment takes the phosphorylation site of Ser47 as the research object.

[0062] (1) Screening possible phosphorylation sites of ATGL

[0063] According to literature reports, mouse ATGL has 10 phosphorylation sites modified, including Ser47, Ser87, Thr101, Thr210, Thr372, Tyr378, Ser393, Ser396, Ser406 and Ser430.

[0064] (2) Construction of expression vector

[0065] In this example, the phosphorylation site of Ser47 was used as the research object, and a plasmid with 10 permanent non-phosphorylation mutations controlled by the CMV promoter (FLAG-FLAG double tag, p9-ATGL-FLAG) was designed as a negative control , the negative control is the expression vector 2, and the ATGL vector map please refer to the attached figure 1 . The base sequence of its amino acid sequence fragment is shown in SEQ ID No.2.

[0066] The 10 permanent non-phosphorylation mutation sites ...

Embodiment 3

[0079] This example provides a method for detecting phosphorylation at a single point of ATGL. The specific method is the same as that in Example 2, and the phosphorylation sites of Ser47, Ser87, Thr101, Thr210, Thr372, Tyr378, Ser393, Ser396, Ser406 and Ser430 were used as the research objects.

[0080] For the above-mentioned 10 phosphorylation sites, expression vectors 1 corresponding to 10 sites to be tested were respectively prepared. Please refer to Table 1 for sequence information.

[0081] Table 1 Sequence information

[0082]

[0083] ATGL10 phosphorylation site plasmid vector 1 was transfected into HL-1 cells for 48 hours to collect protein, and FLAG magnetic beads immunoprecipitation reaction to separate ATGL protein. For results of silver staining and immunoblotting, please refer to the attached image 3 .

[0084]The results of silver staining showed that the FLAG magnetic bead binding protein was mainly ATGL protein with FLAG tag expressed by the expression ...

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Abstract

The invention discloses a method for identifying protein phosphorylation sites, and relates to the field of molecular biology technology. Specifically, the method for identifying protein phosphorylation sites includes separating the protein expressed by expression vector 1 through immunoprecipitation experiments , using phosphorylated antibodies to perform immunoblotting experiments to detect the modification of the phosphorylation site to be tested, the expression vector 1 contains an amino acid sequence fragment with the site to be tested, which is under the condition of no phosphorylated antibody against a single protein site , can also effectively and accurately detect the phosphorylation modification of a single site of the protein, save the cost of preparing phosphorylated antibodies for specific sites, and speed up the research process of protein phosphorylation sites.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for identifying protein phosphorylation sites. Background technique [0002] The phosphorylation and dephosphorylation process of protein regulates important life activities such as cell signal transduction, cell metabolism, cell differentiation and cell growth, and is the molecular switch of cell physiological activities. Different protein kinases can recognize and modify different sites of different proteins, which increases the complexity of the study of phosphorylated proteins, making phosphorylated proteins a hotspot in the study of protein post-translational modification. [0003] use 32 P selectable tagging of phosphorylated proteins is a classic technique in which purified kinases and 32 P for in vitro labeling, or using 32 P-ATP or 32 PO 4 3- (orthophosphate) for in vivo labeling, then the proteins were separated by SDS-PAGE, 2D-GE or thin-layer ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G16B25/00
CPCG01N33/68G16B25/00
Inventor 李凌海
Owner BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV
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