Ubiquitin chain solid phase detection method and application

A detection method and technology for ubiquitin chains, which are applied in a kit for detecting ubiquitin proteins, connect hybrid ubiquitin protein binding domains in series, and detect ubiquitin chains, which can solve the problem of lack, sensitivity, and unbiasedness of ubiquitin chains. The performance and accuracy need to be improved, and the recognition efficiency of ubiquitin antibodies is low, so as to achieve the effect of strong operability, high sensitivity, and low detection limit.

Active Publication Date: 2019-10-08
BEIJING PROTEOME RES CENT
View PDF3 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different spatial topological structures lead to different exposure degrees of ubiquitin epitopes in different types of ubiquitin chains. If the epitopes of ubiquitin molecules are embedded, the recognition efficiency of ubiquitin antibodies will be reduced. The specific manifestation is ubiquitin antibodies There is a preference for the recognition of ubiquitin chains
The resulting problem is that the ubiquitin antibody cannot truly reflect the intensity of the ubiquitination signal in the sample
At present, there is a lack of detection methods that can distinguish between monoubiquitination modification and polyubiquitin chain modification, and the sensitivity, unbiasedness and accuracy of existing ubiquitin antibodies to detect ubiquitin chains need to be improved

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Ubiquitin chain solid phase detection method and application
  • Ubiquitin chain solid phase detection method and application
  • Ubiquitin chain solid phase detection method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Effectiveness and specificity of ThUBD solid-phase recognition of ubiquitinated proteins

[0054] 1. Expression and purification of ThUBD fusion protein

[0055]The expression vector of ThUBD-encoding gene containing GST tag was transformed into Escherichia coli BL21(DE3) (product of Beijing Kangwei Century Biotechnology Co., Ltd., catalog number: CW0808A). Cultivate with LB medium at 37°C until the density is A600 (absorbance value at 600nm) is 0.4-0.6, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5mM, and induce the expression of the fusion protein at 30°C for 4 hours, collect the bacterial solution and centrifuge at 6000rpm, 4°C for 5 minutes to collect the cells.

[0056] The collected cells were resuspended in the lysate (recipe: 1×PBS, pH 7.4, 1mM DTT, 10% glycerol), and the cells were lysed by a sonicator (working for 2 seconds, resting for 4 seconds, sonicating for a total of 30 minutes, with a power of 25% ), and the...

Embodiment 2

[0061] Example 2: The sensitivity of ThUBD solid-phase recognition of ubiquitinated proteins is higher than that of detection methods based on anti-ubiquitin antibodies

[0062] 1. Expression and purification of ThUBD fusion protein

[0063] For the expression and purification methods of ThUBD fusion protein, refer to Example 1.

[0064] 2. Using ThUBD to detect protein ubiquitination in eukaryotic cells

[0065] The 293T cells were cultured and collected, and the total protein of the cells was extracted using the lysate, as described in Example 1. Different amounts of total cellular proteins of 293T cells were separated by 10% SDS-PAGE electrophoresis, transferred to solid-phase NC membranes, and incubated and detected with the method of the present invention and commercial anti-ubiquitin antibodies respectively. The NC membranes used and antibody refer to Example 1. The amount of total protein loaded on 293T cells was increased sequentially, and was set to 1.25, 2.5, 5, 1...

Embodiment 3

[0068] Example 3: Solid-phase recognition and detection of 8 ubiquitin chains by ThUBD

[0069] 1. Expression and purification of ThUBD fusion protein

[0070] The expression and purification method of ThUBD fusion protein, color development method refer to embodiment 1.

[0071] 2. Using ThUBD to detect ubiquitin monomers and di-ubiquitin chains

[0072] The affinity of ThUBD to ubiquitin molecules and diubiquitin chains was tested. Equal mass of ubiquitin and 2-merized K63 chains were incubated with ThUBD at the same time for color detection, and the silver-stained gel image was used to characterize the loading amount. The result is as Figure 4 As shown, in the case of the same loading amount, ThUBD showed a high affinity for ubiquitin chains, but a weak ability to recognize ubiquitin monomers, and there was still no detection signal after long-term exposure, indicating that ThUBD mainly recognized ubiquitin chain, but weakly bound to ubiquitin monomers, consistent with...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
Login to view more

Abstract

The invention relates to a high-efficiency sensitive and unbiased detection method for protein ubiquitin modification in a bio-sample. Artificial synthesis polypeptide formed by two different ubiquitin combination domains in series and including tag protein (GST) in the end N serves as a detection agent. Compared with commercial ubiquitin antibody, the provided detection agent and solid phase detection method can identify 8 different ubiquitin chains formed by ubiquitin molecules in an efficient and unbiased way, the endogenous ubiquitin chain of the bio-sample can be detected without bias, and the detection sensitivity for ubiquitin protein can be improved substantially.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for detecting ubiquitin chains; [0002] The invention also relates to the use of tandem hybrid ubiquitin binding domains (ThUBDA); [0003] The present invention also relates to a kit for detecting ubiquitin protein. Background technique [0004] Ubiquitin is a small protein with 76 amino acids, named for its ubiquitous presence and high conservation in eukaryotic cells. Ubiquitin-proteasome system (Ubiquitin Proteasome System, UPS) is one of the important protein-specific degradation pathways in eukaryotes. Studies have shown that, in addition to mediating the specific degradation of proteins, ubiquitination modification, as an important regulatory signal, also participates in almost all life activities including cellular immune response, DNA damage repair and cell apoptosis. The dysregulation of key nodes of ubiquitination modification is closely related to the occurrenc...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543G01N30/02
CPCG01N33/68G01N33/6848G01N33/54306G01N30/02
Inventor 李衍常徐平肖伟弟高媛常蕾高慧英
Owner BEIJING PROTEOME RES CENT
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap