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Functionalized temperature-sensitive polymer and its preparation method and application in (ptm) protein detection

A temperature-sensitive, polymer-based technology, applied in the field of analysis, can solve the problems of limiting WB detection sensitivity and quantitative accuracy, and achieve the effect of improving binding ability

Active Publication Date: 2019-11-26
ACAD OF MILITARY SCI ACAD OF MILITARY MEDICAL SCI BEIJING INST OF LIFEOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during incubation, the antibody immobilized on the insoluble carrier forms a heterogeneous reaction system with the target protein on the PVDF membrane, resulting in significant interfacial mass transfer resistance, nonlinear reaction kinetics and steric hindrance, thus limiting the detection sensitivity of WB and quantitative accuracy

Method used

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  • Functionalized temperature-sensitive polymer and its preparation method and application in (ptm) protein detection
  • Functionalized temperature-sensitive polymer and its preparation method and application in (ptm) protein detection
  • Functionalized temperature-sensitive polymer and its preparation method and application in (ptm) protein detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1, preparation and performance test of functionalized temperature-sensitive polymer

[0076] 1. Preparation

[0077] according to figure 1 The synthetic route shown prepares the functionalized temperature-sensitive polymer, and the specific steps are as follows

[0078] (1) Synthesis of temperature-sensitive polymers: In the presence of initiators, temperature-sensitive monomers and functional monomers undergo radical polymerization reactions to obtain temperature-sensitive polymers Poly(NIPAM-co -AA). details as follows:

[0079] Weigh 0.8g of N-isopropylacrylamide into a 50mL round bottom flask, add 20mL of deionized water to fully dissolve it, add 0.12mL of acrylic acid, stir and mix well, and adjust the pH to 7.0 with 1M NaOH. Then pass N 2 Stir magnetically for 1 h to fully remove O 2 , after 1h, remove N 2 device, seal the round bottom flask, put a stopper on the mouth of the bottle and wrap it with a parafilm, and heat up to 70°C. Weigh 80 mg o...

Embodiment 2

[0095] Example 2. Detection of azide-labeled proteins by functionalized temperature-sensitive polymers

[0096] according to Figure 6 The flow chart shown uses the functionalized temperature-sensitive polymer prepared in Example 1 to detect azide-labeled proteins, and the specific steps are as follows:

[0097] (1) Azide labeling of proteins or their PTMs:

[0098] Labeling by cell metabolism; or using in vitro enzyme-catalyzed labeling.

[0099] Cell metabolism labeling: HeLa cells were cultured in a 37°C incubator. After the density was appropriate, the culture medium was poured out, washed 3 times with PBS, and the cells were cultured in 10mL DMEM containing 10% fetal bovine serum and 1% penicillin and streptomycin mixture. Azide metabolism reagents were added to the medium to make the final concentration 200 μM. After 24 hours of metabolic culture, 10 mL PBS was used to wash 3 times to remove excess azide metabolism reagents. Cells were collected and proteins were extra...

Embodiment 3

[0107] Example 3. Detection of Azide-labeled O-GlcNAc Modified Standard Proteins Using Functionalized Temperature Sensitive Polymers

[0108] (1) According to the in vitro enzyme-catalyzed method described in Example 2, the standard O-GlcNAc protein α-crystallin was azide-labeled. After azide labeling and purification of the α-crystallin standard protein, it was dissolved in 20mM Hepes buffer solution (pH 7.9) and stored at -80°C for future use.

[0109] (2) Using the SDS-PAGE protein separation, membrane transfer and WB analysis strategies based on functionalized temperature-sensitive polymers described in Example 2, WB analysis was performed on azide-labeled α-crystallin proteins with different loading amounts, The result is as Figure 7 as shown in a. As the loading amount of azide-labeled α-crystallin decreased from 2 μg to 0.01 μg, the gray scale / intensity of its bands in WB detection also weakened, with a good signal response, indicating that this strategy can be used ...

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Abstract

The invention discloses a functionalized temperature-sensitive polymer, a preparation method of same, and an application of same in (PTM) protein detection. The structure formula of the functionalizedtemperature-sensitive polymer is represented as the formula I, wherein R is CH2, r is 0 or 1, m is any natural number from 20 to 750, n is any natural number from 110-1120, and the structure in the specification is horse radish peroxidase unit. The functionalized temperature-sensitive polymer is a linear polymer, which is produced through a free radical polymerization reaction and has a large number of modificable loci. After functional modification, the temperature-sensitive polymer having a plurality of HRP and a large number of triphenyl phosphine groups can be produced. Compared with an antibody strategy in which only single HRP is carried in the prior art, increasing of numbers of the HRP and the triphenyl phosphine groups can effectively increase the combination capability between the polymer and azide-labeled target proteins and signal amplification effect of chemical-luminescence.

Description

technical field [0001] The invention relates to a functionalized temperature-sensitive polymer, a preparation method thereof and an application in (PTM) protein detection, belonging to the field of analysis technology. Background technique [0002] Western Blot (WB) is an analytical technique for detecting protein expression and distribution in complex biological samples. It has been widely used in biological research for decades and can perform qualitative and semi-quantitative analysis. It is a modern biomedical A powerful tool for research in fields such as biochemistry and immunogenetics. In this technique, the target protein is separated by SDS-PAGE and transferred to a PVDF membrane, incubated with an antibody to identify the target protein, and undergoes a chemiluminescence reaction with the substrate by an antibody ligase (such as horseradish peroxidase (HRP)) , so as to generate color signals to achieve the purpose of detection. The traditional WB technology is ba...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08F220/54C08F220/06C08F8/32G01N33/68G01N21/76
CPCC08F8/32C08F220/54G01N21/76G01N33/68C08F220/06
Inventor 钱小红秦伟捷刘彤张万军
Owner ACAD OF MILITARY SCI ACAD OF MILITARY MEDICAL SCI BEIJING INST OF LIFEOMICS
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