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Method for detectingprotein interaction by co-immunoprecipitation on basis of two-color fluorescent tag proteins GFP and mCherry

A technology of co-immunoprecipitation and labeling proteins, which is applied in material analysis by optical means, fluorescence/phosphorescence, measuring devices, etc. It can solve problems such as difficult real-time monitoring of target protein full expression, time-consuming, and difficult results.

Active Publication Date: 2019-11-15
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After coupling the above-mentioned tagged proteins, the protein expression can only be verified by Western Blot, which is cumbersome, time-consuming, highly technically demanding, and the results are not easy to stabilize. It is also difficult to monitor the two targets in real time. Whether the protein is fully expressed in foreign cells
There are many unstable factors in the experiment, and it is difficult to draw conclusions for negative results

Method used

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  • Method for detectingprotein interaction by co-immunoprecipitation on basis of two-color fluorescent tag proteins GFP and mCherry
  • Method for detectingprotein interaction by co-immunoprecipitation on basis of two-color fluorescent tag proteins GFP and mCherry
  • Method for detectingprotein interaction by co-immunoprecipitation on basis of two-color fluorescent tag proteins GFP and mCherry

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Experimental program
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Embodiment 1

[0055] A method for detecting protein interaction by co-immunoprecipitation based on two-color fluorescent label protein GFP and mCherry, comprising the following steps:

[0056] (1) Construct vectors containing target genes to be verified for interaction

[0057] The In-Fusion cloning reaction system was established, and the two target genes to be verified for interaction were homologously recombined into the fluorescent transient expression vectors pBinplus-GFP (CN201711362357.4) and pBinplus-mCherry (Li et al., 2019), the plasmid was extracted and transformed into Agrobacterium tumefaciens GV3101 by electric shock (Li et al., 2019).

[0058] (2) Instantly transform tobacco leaves after mixing the above-mentioned Agrobacterium infection solution in equal proportions

[0059] Dip the above-mentioned GV3101 bacterial liquid with correct bacterial inspection into the 4~6ml LB liquid medium containing 50ug / ml kanamycin and 50ug / ml rifampicin resistance respectively, and shake c...

Embodiment 2

[0068] The exogenous Cry1Ab / c protein (Tu et al., 1998) expressed in the insect-resistant transgenic rice Huahui No. 1 (Tu et al., 1998) and three kinds of endogenous rice protein calmodulin binding transcription factor CAMTAs of different molecular weights were used in the specific method of Example 1 (Gene ID: XM_015758912.2), 3-deoxy-D-arabinose heptulose-7-phosphate DAHP (Gene ID: XM_015790892.1), histone lysine methyltransferase HKMTs (Gene ID : XM_015792076.2) to detect protein interaction by co-immunoprecipitation, the above three proteins are rice endogenous proteins that can interact with exogenous Cry1Ab / c protein in yeast two-hybrid primary screening.

[0069] The specific construction method is as follows: refer to the method in Example 1 to establish the In-Fusion cloning reaction system, homologously recombine the cry1Ab / c gene into the N-terminus of the fluorescent transient expression vector pBinplus-GFP through the endonuclease BamH, and respectively integrate ...

Embodiment 3

[0076] Example 3 Verify the validity of the above co-immunoprecipitation results by bimolecular fluorescence complementary technology

[0077] According to the method of Example 1, the In-Fusion cloning reaction system was established, and the cry1Ab / c gene was homologously recombined into the C-terminus of the bimolecular fluorescence complementary expression vector PCV-cYFP (Hu et al., 2015) by endonuclease BamH , homologous recombination of CAMTAs, DAHP and HKMTs genes into the C-terminus of the expression vector PCV-nYFP (Hu et al., 2015). The homologous recombination primers are shown in Table 3. After extracting the plasmid, electric shock transformed Agrobacterium GV3101 to obtain exogenous cry1Ab GV3101 infection liquid containing c-cYFP gene, GV3101 infection liquid containing CAMTAs-nYFP gene, GV3101 infection liquid containing DAHP-nYFP gene and GV3101 infection liquid containing HKMTs-nYFP gene.

[0078] The GV3101 infection solution containing the exogenous cry1Ab...

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Abstract

The invention discloses a method for detectingprotein interaction by co-immunoprecipitation on the basis of two-color fluorescent tag proteins GFP and mCherry. The method comprises steps as follows: target genes are constructed to N ends of expression vectors respectively, agrobacteria are transformed after plasmid is extracted, agrobacterium infection liquid co-injection model plant is prepared,a co-injection tobacco leaf protein is extracted, target proteins with GFP tags are enriched with GFP-Trap A beads, meanwhile, other target proteins with mCherry tagsare immunized, and finally, expression and interaction conditions of the target proteins before immunization and after immunization are detected by the GFP and mCherry tag antibodies respectively. According to the method, while real-time fluorescent visual monitoring for expression and co-localization of the target proteins in living cells is realized,the protein interaction can be directly and effectively verified through the co-immunoprecipitationby selecting the stage of the highest expression quantity in real time, the success rate of protein interaction verification is greatly increased, and time and economic cost is substantially reduced.

Description

[0001] Technical Field The present invention relates to the application of protein interaction technology, and in particular to a method for detecting protein interaction by co-immunoprecipitation based on two-color fluorescent label protein GFP and mCherry. Background technique [0002] Protein is the carrier of life activities and the executor of functions. Proteins form a protein interaction network through the interaction with each other to participate in various aspects of life processes such as biological signal transmission, gene expression regulation, energy and material metabolism, and cell cycle regulation. . Through the comprehensive application of yeast two-hybrid, co-immunoprecipitation and bimolecular fluorescence complementation, people study the authenticity of protein interaction, and then explore the deep molecular mechanism of related physiological phenotypes. Among them, the co-immunoprecipitation technique is a classic method for studying protein interacti...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/533G01N33/541G01N33/58G01N21/64
CPCG01N33/6845G01N33/533G01N33/541G01N33/582G01N21/6458
Inventor 付健美施雨纪锐方继朝
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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