[go: up one dir, main page]

WO2025080789A1 - Procédés pour caractériser les historiques compositionnels et fonctionnels de protéines cellulaires et complexes macromoléculaires associés dans l'espace et le temps - Google Patents

Procédés pour caractériser les historiques compositionnels et fonctionnels de protéines cellulaires et complexes macromoléculaires associés dans l'espace et le temps Download PDF

Info

Publication number
WO2025080789A1
WO2025080789A1 PCT/US2024/050705 US2024050705W WO2025080789A1 WO 2025080789 A1 WO2025080789 A1 WO 2025080789A1 US 2024050705 W US2024050705 W US 2024050705W WO 2025080789 A1 WO2025080789 A1 WO 2025080789A1
Authority
WO
WIPO (PCT)
Prior art keywords
ligand
proteins
tracking
complexes
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/050705
Other languages
English (en)
Inventor
Jordy F. BOTELLO
Clifford P. Brangwynne
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Princeton University
Original Assignee
Princeton University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Princeton University filed Critical Princeton University
Publication of WO2025080789A1 publication Critical patent/WO2025080789A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5035—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on sub-cellular localization
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the analyses may include (i) tracking the plurality of endogenous proteins and any associated macromolecular complexes using a ligand that binds to the fusion proteins; (ii) determining composition and function of the plurality of endogenous proteins and/or any associated macromolecular complexes; and/or (iii) removing a ligand from a cell feeding media, where the cell feeding media is used to perform a chase in a pulse-chase analysis, as new proteins won’t be able to bind the (now removed) ligand.
  • kits may be provided.
  • the kit may include a universal genetic tag; and a first ligand, the first ligand (such as a halo tag ligand) being a ligand with a developed immunopurification capability and a known imaging capability.
  • Figure 3 is a schematic illustration of a nucleotide sequence expressing a protein construct.
  • Figure 7 is a schematic illustration of pulse-chase experiments using a haloalkane dehalogenase tag.
  • Figure 19 is a plot of nascent large subunit (LSU) ribosomal proteins enriched over time, as a percentage of steady state.
  • LSU large subunit
  • Figures 21A and 21B are plots of eukaryotic translation initiation factor 3 subunits enriched over time, as a percentage of steady state.
  • Figure 22 is a plot of eukaryotic translation initiation factor 4 subunits enriched over time, as a percentage of steady state.
  • Figure 24 is a diagram that illustrates recording nucleolar pre-60S assembly, as well as sample images captured during the process.
  • the use of a universal genetic tag allows for a high degree of modularity that would make this process accessible to most of the cellular proteome.
  • This process has the potential to opening the study of an understudied dimension of cellular protein function - molecular age.
  • this strategy could also be used to characterize the assembly of nascent macromolecular machines, such as ribosomes or spliceosomes.
  • the first ligand (224) would include a dye or marker (such as a tetramethylrhodamine (TMR) dye) that allows the components that the first ligand covalently binds with to be tracked.
  • TMR tetramethylrhodamine
  • the first ligand is replaced with a blocking ligand (230).
  • the blocking ligand may be, e.g., a ligand that only has the, e.g., binding portion (228) of the first ligand, that binds to the universal genetic tag.
  • the blocking ligand may include a fluorescent portion (228), but emit a different wavelength from that used by the first ligand.
  • the fusion proteins may include a cleavage tag (221) disposed between the protein of interest (e.g., endogenous protein (210) and the universal genetic tag (222).
  • the cleavage tag (221) may be any appropriate known cleavage tag.
  • Such cleavage tags include, e.g., selfcleaving tags, Human Rhinovirus 3C Protease (3C/PreScission), Enterokinase (EKT), Factor Xa (FXa), Tobacco Etch Virus Protease (TEV), or Thrombin (Thr)cleavage tags
  • a protein of interest (POI) (614) is fused to a haloalkane dehalogenase tag (610), where the haloalkane dehalogenase tag includes a cleavage site (such as a TEV cleavage site).
  • Haloalkane dehalogenase tags bind haloalkane ligands irreversibly, the haloalkane ligand forming a covalent bond (630) with the haloalkane dehalogenase tag when the halogen (here, a chlorine ion) is removed.
  • a nucleic acid sequence (300), such as an isolated polynucleotide sequence, may be provided, where a polynucleotide sequence (320) configured to express the fusion protein (220) may be placed under the control of a promoter (310), such as an endogenous promoter. Any endogenous promoter may be utilized.
  • the universal genetic tag may be any appropriate universal genetic tag.
  • a universal genetic tag is a molecular marker that can be used to label and track proteins across different systems, organisms, or experimental conditions. The goal of such tags is to provide a consistent and versatile method for monitoring protein expression, localization, or interactions regardless of the specific context in which they are used. Such tags are considered “universal” because they can be applied to various experimental systems and provide reliable and reproducible results. They facilitate protein manipulation and analysis across different research contexts.
  • HALOTAG® protein tag a protein tag derived from the Haloalkane Dehalogenase enzyme. It covalently binds to a synthetic ligand or substrate that is conjugated to a fluorophore or other imaging probe.
  • fluorophore-conjugated HALOTAG® ligands are available, enabling live-cell imaging and tracking of proteins with high specificity.
  • CLIP-tag a variant of the O6-alkylguanine DNA alkyltransferase, optimized for binding to different synthetic ligands than SNAP-tag. Similar to SNAP-tag, CLIP-tag ligands are available with various fluorophores, enabling fluorescent imaging of proteins in live cells.
  • tetramethylrhodamine-bound HaloTag-fusion complexes may be immunopurified by producing superparamagnetic beads covalently fused to high affinity anti-tetramethylrhodamine antibodies. These allow immobilization of complexes in these beads, performing washes and eluting off the beads either by denaturation or protease cleavage (TEV, etc).
  • TSV protease cleavage
  • the method may include analyzing (140) the proteins and/or recording (150) a compositional and functional history of the plurality of endogenous proteins in space and time.
  • analysis e.g., part of the analyzing (140) step
  • the analysis step may include tracking (142) the plurality of endogenous proteins and any associated macromolecular complexes. Tracking may be accomplished via live-cell confocal microscopy.
  • Functional Genomics e.g., gene expression studies to analyze how the protein’s gene is regulated under various conditions using techniques like quantitative PCR or RNA sequencing, and/or proteomics to study the protein’s interactions with other proteins, lipids, or nucleic acids within the cell).
  • the analysis step may include removing (146) a ligand from cell feeding media, and may include performing apulse-chase analysis using the plurality of endogenous proteins. Such techniques are well understood in the art.
  • first ligands are used to label preexisting I parental population to saturation.
  • a second ligand can be used to label nascent populations continuously.
  • composition and function are determined by immunopurification followed by quantitative mass spectrometry and/or genomic analysis.
  • the method (100) may also include using the fusion proteins to identify agent that specifically targets an old or a young protein.
  • the term “young protein” and “old protein” refers to the relative times in which a protein is synthesized during a process.
  • a young protein may be considered on a relative basis as, e.g., one of the top 50%, top 33%, top 25%, or top 10% most recently synthesized fusion proteins existing at a location at any point in time.
  • a young protein may be considered on an absolute basis as, e.g., a labelled protein that was synthesized less than, e g., a predetermined threshold of time such as 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 1 minute, 30 seconds, 15 seconds, or 10 seconds in the past.
  • a predetermined threshold of time such as 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 1 minute, 30 seconds, 15 seconds, or 10 seconds in the past.
  • the method may therefore include providing (410) at least one of the fusion proteins to each of a plurality of containers.
  • the method may include exposing (420) each container to one or more chemical or biological agents.
  • the method may include identifying (430) a chemical or biological agent that specifically targets an old or a young protein by performing the at least one analysis (140).
  • a camera (520) may be disposed above the containers, and at least one lens (522) may be used to allow the camera to monitor the proteins within the container(s).
  • One or more light sources (530) may be arranged to irradiate the labelled proteins, to enable the camerato capture details as desired. For example, if a fluorescently labelled protein is within a container, the light source may be configured to emit a wavelength of light that can activate the fluorescent tag, allowing the camera to capture the light emitted from the fluorescent tag.
  • the one or more lenses (522) may include a filter (524) for aiding in the detection of the protein tags.
  • the camera and/or the light source may be coupled to a controller (540).
  • the controller may include one or more processors (542), anon-transitory storage device (544), and memory (546).
  • a display (550) may be coupled to the camera and/or the controller.
  • the controller may be configured to control the light source and/or the camera, and the controller may be configured to store data (including one or more images I video) captured by the camera.
  • the controller is configured to display the image(s) or video captured by the camera.
  • the containers may be stationary relative to the camera. In some embodiments, the containers may move relative to the camera.
  • FIG. 9 provides a schematic summary' of the toolkit and strategy disclosed herein.
  • the experimental components include bio-orthogonal fluorescent ligands, universal protein tags, such as HALOTAG® protein tags, can be used for controlled covalent labelling, and the use of ribosome homozygous tagging (RHT).
  • RHT ribosome homozygous tagging
  • FIGS. 13-17 showcase visualization of nascent ribosomes, their quantification, and proteomics data demonstrating the high quality of the purification possible using the disclosed approach.
  • cytoplasmic 40S maturation and function was considered using the basic process outlined in FIG. 13, including analyzing images captured, e.g., at 0, 2, 4, 8, 24, and additional images captured until the process reached a steady state.
  • FIG. 14 shows the evolution of nascent 40S labelling over time, as a percent of the final steady state.
  • FIG. 15 shows a graph of fold change vs adjusted p-value for comparing a RPS 17-HaloTag fusion (with a tetramethylrhodamine (TMR) dye) vs just the HaloTag(TMR).
  • TMR tetramethylrhodamine
  • a preferred approach for the disclosed methodology utilizes this tetramethyrhodamine ligand to allow purification and imagine.
  • FIG. 16 shows a barchart of detected known sequences, using the disclosed approach, for cytoplasmic 40S immunoprecipitation (IP).
  • FIG. 17 shows a visualization of the gene ontology for cytoplasmic 40S immunoprecipitation (IP).
  • FIGS. 18-23 demonstrate that the disclosed approach can indeed resolve the spatiotemporal compositional dynamics of ribosomes.
  • FIGS. 18-20 relate to assembly of ribosomes
  • FIGS. 21A-23 relate to function of ribosomes.
  • FIG. 18 illustrates the assembly of small subunits of ribosomal proteins over time
  • FIG. 19 illustrates the assembly of large subunits of ribosomal proteins over time.
  • FIG. 20 illustrates change in assembly factors (including RRP12, RIOK1, PNO1, LTV1, NOB1, RIOK2, and BYSL) over time.
  • FIGS. 21A-21B show expression of some subunits of eukaryotic initiation factor 3 (eIF3) over time.
  • eIF3 eukaryotic initiation factor 3
  • FIG. 25 shows core (e.g., fibrillar center) and rim (e.g., granular component) enrichment over time for large ribosomal subunit proteins.
  • FIGS. 26-27 are charts tracking pre-60S ribosomal proteins (26) assembly factors (27) over time.
  • FIG. 28 tracks nucleolar proteins over time.
  • FIG. 29 shows the early depletion of certain nucleolar proteins, and when the depletion occurs. As seen, it is clear that the two proteins shown are generally not depleting at all around four hours into the process, whereupon both proteins suddenly get substantially depleted by hour six, and clearly almost entirely depleted by hour eight. Similarly, FIG.
  • snoRNP small nucleolar ribonucleoproteins

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Est divulgué un procédé de caractérisation d'historiques compositionnels et fonctionnels de protéines cellulaires et de leurs complexes moléculaires associés dans l'espace et le temps. Le procédé peut comprendre la formation de protéines de fusion par fusion d'une étiquette génétique universelle à chaque protéine d'une pluralité de protéines endogènes. Les protéines de fusion peuvent être marquées de manière conditionnelle avec un premier ligand. Le premier ligand peut être un ligand présentant une capacité d'immunopurification développée et une capacité d'imagerie connue. Le procédé peut comprendre l'expression des protéines de fusion et la réalisation d'au moins une analyse. Les analyses peuvent comprendre (i) le suivi de la pluralité de protéines endogènes et des éventuels complexes macromoléculaires associés ; (ii) la détermination d'une composition et d'une fonction de la pluralité de protéines endogènes et/ou des éventuels complexes macromoléculaires associés ; et/ou (iii) l'élimination d'un ligand et la réalisation d'une analyse pulse-chase.
PCT/US2024/050705 2023-10-10 2024-10-10 Procédés pour caractériser les historiques compositionnels et fonctionnels de protéines cellulaires et complexes macromoléculaires associés dans l'espace et le temps Pending WO2025080789A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363543329P 2023-10-10 2023-10-10
US63/543,329 2023-10-10

Publications (1)

Publication Number Publication Date
WO2025080789A1 true WO2025080789A1 (fr) 2025-04-17

Family

ID=95396356

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/050705 Pending WO2025080789A1 (fr) 2023-10-10 2024-10-10 Procédés pour caractériser les historiques compositionnels et fonctionnels de protéines cellulaires et complexes macromoléculaires associés dans l'espace et le temps

Country Status (1)

Country Link
WO (1) WO2025080789A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150125904A1 (en) * 2012-03-30 2015-05-07 Massachusetts Institute Of Technology Probe incorporation mediated by enzymes
US20190365818A1 (en) * 2017-02-09 2019-12-05 Allen Institute Genetically-tagged stem cell lines and methods of use
US20230137943A1 (en) * 2019-09-20 2023-05-04 The University Of Chicago Photoproximity profiling of protein-protein interactions in cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150125904A1 (en) * 2012-03-30 2015-05-07 Massachusetts Institute Of Technology Probe incorporation mediated by enzymes
US20190365818A1 (en) * 2017-02-09 2019-12-05 Allen Institute Genetically-tagged stem cell lines and methods of use
US20230137943A1 (en) * 2019-09-20 2023-05-04 The University Of Chicago Photoproximity profiling of protein-protein interactions in cells

Similar Documents

Publication Publication Date Title
Sridharan et al. Systematic discovery of biomolecular condensate-specific protein phosphorylation
Lalonde et al. Molecular and cellular approaches for the detection of protein–protein interactions: latest techniques and current limitations
Reinke et al. In vivo mapping of tissue-and subcellular-specific proteomes in Caenorhabditis elegans
Phizicky et al. Protein analysis on a proteomic scale
Kerppola Visualization of molecular interactions using bimolecular fluorescence complementation analysis: characteristics of protein fragment complementation
Hung et al. Proteomic mapping of the human mitochondrial intermembrane space in live cells via ratiometric APEX tagging
Berggård et al. Methods for the detection and analysis of protein–protein interactions
Yarrow et al. Phenotypic screening of small molecule libraries by high throughput cell imaging
JP2000509827A (ja) 細胞に基づくスクリーニングシステム
US20170292958A1 (en) Methods for proteomic profiling using non-natural amino acids
Paul et al. Imaging the future: the emerging era of single‐cell spatial proteomics
WO2022242895A1 (fr) Marqueur, procédé et dispositif d'analyse d'un échantillon biologique
Demaurex Calcium measurements in organelles with Ca2+-sensitive fluorescent proteins
EP4092414B1 (fr) Connecteur, marqueur et procédé pour analyser des échantillons biologiques
Rimbault et al. Engineering paralog-specific PSD-95 recombinant binders as minimally interfering multimodal probes for advanced imaging techniques
Segers‐Nolten et al. Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes
CN108519484A (zh) 利用自连接标签的双分子荧光互补技术成像和追踪活细胞内的蛋白质相互作用
US20230258628A1 (en) Connector, marker and method for analysing biological samples
Damelin et al. Nuclear protein transport
Presley Imaging the secretory pathway: the past and future impact of live cell optical techniques
Velay et al. MoBiFC: development of a modular bimolecular fluorescence complementation toolkit for the analysis of chloroplast protein–protein interactions
WO2025080789A1 (fr) Procédés pour caractériser les historiques compositionnels et fonctionnels de protéines cellulaires et complexes macromoléculaires associés dans l'espace et le temps
EP3172563B1 (fr) Procédés pour détecter des interactions dans des cellules eucaryotes au moyen de structures de microtubules et dynamiques
Huang et al. Detecting protein− protein interaction during liquid− liquid phase separation using fluorogenic protein sensors
JP4748719B2 (ja) 生細胞内の特定タンパク質の定量方法および標準蛍光マイクロビーズの作製方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24877981

Country of ref document: EP

Kind code of ref document: A1