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A method for the detection of protein kinases based on surface plasmon resonance

A surface plasmon and protein kinase technology, applied in the field of chemistry, can solve the problems of poor protein non-specific ability, complex preparation process and high cost, and achieve the effects of preventing non-specific adsorption, simple preparation and low cost

Inactive Publication Date: 2018-07-10
ANYANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these self-assembled membranes also have some disadvantages, such as: complex preparation process, high cost, poor ability to prevent non-specificity of proteins, etc.

Method used

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  • A method for the detection of protein kinases based on surface plasmon resonance
  • A method for the detection of protein kinases based on surface plasmon resonance
  • A method for the detection of protein kinases based on surface plasmon resonance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] The preparation of SPR sensor chip, the steps are as follows:

[0017] (1) The gold film with a thickness of 50 nm was purchased from Biosensing Instrument Company, USA. The basic composition of the company's gold film is to coat a 18 mm × 18 mm glass slide with a 2 nm thick Cr metal film to enhance the bonding force between gold and glass, and then coat it with a 50 nm thick Cr metal film. Gold as the sensing layer;

[0018] (2) The gold film was soaked in absolute ethanol for 10 minutes before use, ultrasonicated for 5 minutes, rinsed with secondary water, and cleaned with N 2 blow dry;

[0019] (3) After cleaning, use H 2The flame is annealed on the surface of the gold sheet, preheated first, and then at an angle of 45 degrees H 2 Flame uniform annealing for 30 seconds to clean the surface and eliminate non-specific adsorption;

[0020] (4) Peptide P1 was dissolved in secondary water to 1 mM, then diluted to 10 µM with 10 mM PBS solution of pH 7.2, and polypepti...

Embodiment 2

[0024] The specific and non-specific effects of protein kinase A on the surface of the gold film in Example 1, the steps are as follows:

[0025] (1) Protein kinase A was diluted with 10 mM pH 7.2 PBS buffer;

[0026] (2) Place the prepared P1 / P2 modified gold film on the SPR instrument, when the baseline is stable, flow inject 25 nM protein kinase A solution, and record the SPR response signal until it becomes stable. The carrier liquid is 10 mM PBS buffer solution, pH 7.2, and the flow rate is 10 µL / min;

[0027] (3) Place the prepared P2-modified gold film on the SPR instrument. When the baseline is stable, inject 25 nM protein kinase A solution and record the SPR response signal until it stabilizes. The carrier liquid is 10 mM PBS buffer solution, pH 7.2, and the flow rate is 10 µL / min;

[0028] (4) Compare the SPR response signals of the protein kinase A solution injected on the surface of different gold membranes, such as figure 2 Shown: protein kinase A produced a s...

Embodiment 3

[0029] Embodiment 3: the regeneration of sensor chip, the steps are as follows:

[0030] (1) Dilute protein kinase A with 10 mM, pH 7.2 PBS buffer;

[0031] (2) Place the prepared P1 / P2 modified gold film on the SPR instrument, when the baseline is stable, flow inject 25 nM protein kinase A solution, and record the SPR response signal until it becomes stable. The carrier liquid is 10 mM PBS buffer solution, pH 7.2, and the flow rate is 10 µL / min;

[0032] (3) Inject 50 mM NaOH solution into the detection channel of step (2) at a flow rate of 10 µL / min to elute the protein kinase A bound to the surface of the sensing gold membrane, allowing the sensing gold membrane to regenerate until the SPR signal Reach the size before the injection of protein kinase A to prepare for the detection of the next sample;

[0033] (4) Inject the same concentration of protein kinase A solution as in step (2) onto the surface obtained in step (3), record the SPR signal until the baseline is stabl...

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Abstract

A method for detecting protein kinase based on surface plasmon resonance comprises the following steps: A, a gold film is used as a sensing layer, the sensing layer, first, is soaked with ethanol, then ultrasonically cleaned, H2 flame is used for annealing on the surface of the gold film; B, the gold film processed by the step A is soaked in a polypeptide P1 solution with an amino acid sequence shown as SEQIDNO:1, then soaked with the gold film in a polypeptide P2 solution with an amino acid sequence shown as SEQIDNO:2, and blown to dry with N2, and sealed storage of the prepared polypeptide P1 and P2-modified sensing gold film is performed at-18 DEG C for standby use; and C, the polypeptide P1 and P2-modified sensing gold film prepared by the step B is put on a SPR (surface plasmon resonance) apparatus, protein kinase flow injection is performed on the sensing gold film, when the sensing gold film is bonded with polypeptides on the surface of the gold film, a new resonance angle is produced, and the size of the resonance angle is a SPR response signal.

Description

technical field [0001] The invention relates to a surface technology for protein kinase detection, in particular to a method for detecting protein kinase based on surface plasmon resonance, which belongs to the field of chemistry. Background technique [0002] Protein phosphorylation plays a decisive role in cell proliferation and differentiation, information transmission between cells, and gene expression. Protein kinase is an enzyme that catalyzes the phosphorylation of proteins. Studies have shown that the occurrence of some major diseases (such as diabetes, Alzheimer's disease, cancer, metabolic syndrome, etc.) is closely related to changes in the content and activity of protein kinases. Therefore, the detection of protein kinase is of great significance in the diagnosis and treatment research of these major diseases. At present, the methods used to detect protein kinases mainly include radioisotope method, fluorescence method, electrochemistry and mass spectrometry, e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/552G01N33/68
CPCG01N21/553G01N33/68
Inventor 刘林夏宁孙婷王薪
Owner ANYANG NORMAL UNIV
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