Source 1primary paper2014PLoS ONE
Toolkit/split PAmCherry1 BiFC probe
split PAmCherry1 BiFC probe
Also known as: PAmCherry1 BiFC, PAmCherry1 split between residues 159 and 160
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
We demonstrated that PAmCherry1, a photoactivatable fluorescent protein commonly used for PALM, can be used as a BiFC probe when split between residues 159 and 160 into two fragments.
Usefulness & Problems
Why this is useful
This construct pattern uses PAmCherry1 split into two fragments so fluorescence is reconstituted upon protein-protein interaction and can then be localized by PALM.; detecting protein-protein interactions with BiFC; enabling PALM localization of complemented interacting complexes
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This construct pattern uses PAmCherry1 split into two fragments so fluorescence is reconstituted upon protein-protein interaction and can then be localized by PALM.
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detecting protein-protein interactions with BiFC
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enabling PALM localization of complemented interacting complexes
Problem solved
It supplies a BiFC probe that retains PALM-compatible photophysical properties after complementation.; provides a photoactivatable BiFC probe compatible with PALM-based super-resolution imaging
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It supplies a BiFC probe that retains PALM-compatible photophysical properties after complementation.
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provides a photoactivatable BiFC probe compatible with PALM-based super-resolution imaging
Problem links
provides a photoactivatable BiFC probe compatible with PALM-based super-resolution imaging
LiteratureIt supplies a BiFC probe that retains PALM-compatible photophysical properties after complementation.
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It supplies a BiFC probe that retains PALM-compatible photophysical properties after complementation.
Published Workflows
Objective: Develop and apply a combined BiFC and PALM method to visualize protein-protein interactions in cells with nanometer spatial resolution and single-molecule sensitivity.
Why it works: The workflow works by making protein-protein interactions selectively generate a reconstituted photoactivatable fluorophore, allowing only interacting complexes to be localized by PALM with high spatial precision.
fluorescence complementation upon protein-protein interactionphotoactivation-based single-molecule localizationbimolecular fluorescence complementationphotoactivated localization microscopy
Stages
- 1.BiFC probe design(library_design)
The method requires a photoactivatable fluorescent protein that can be split for complementation without losing PALM-relevant properties.
Selection: Identify a split configuration of PAmCherry1 that can function as a BiFC probe.
- 2.Probe performance characterization(functional_characterization)
The combined method depends on the complemented fluorophore remaining selective and PALM-compatible after BiFC reconstitution.
Selection: Assess specificity, efficiency, spontaneous reconstitution background, and retained photophysical properties of complemented split PAmCherry1.
- 3.Cellular application to Ras/Raf complexes(confirmatory_validation)
A cellular demonstration is needed to show that the engineered probe and combined imaging method can reveal biologically informative PPI organization and dynamics.
Selection: Apply BiFC-PALM to a biologically relevant protein interaction pair and test whether nanoscale organization and dynamics can be resolved in cells.
Steps
- 1.Split PAmCherry1 between residues 159 and 160engineered BiFC probe design
Create a photoactivatable fluorescent protein complementation probe compatible with PALM.
A split fluorophore design is required before testing whether complementation can support selective super-resolution localization of PPIs.
- 2.Test complemented split PAmCherry1 for specificity, efficiency, background, and PALM-compatible photophysicsprobe under evaluation
Determine whether the split probe can detect PPIs specifically and still support PALM localization after reconstitution.
The probe must be validated for low background and retained photophysical performance before being trusted in a biological application.
- 3.Apply BiFC-PALM to KRas G12D and CRaf RBD complexes on the cell membraneimaging method used for biological application
Demonstrate that the method can resolve nanoscale organization and dynamics of a specific protein interaction in cells.
After probe validation, a cellular use case is needed to show biological utility and the kind of insight the method can provide beyond conventional approaches.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A reusable architecture pattern for arranging parts into an engineered system.
Mechanisms
bimolecular fluorescence complementationphotoactivationsingle-molecule localization microscopyTechniques
No technique tags yet.
Target processes
localizationInput: Light
Implementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationimplementation constraint: spectral hardware requirementoperating role: sensorswitch architecture: split
It requires the photoactivatable fluorescent protein PAmCherry1, a split design at residues 159 and 160, and fusion of the fragments to interacting protein partners.; must be split between residues 159 and 160 into two fragments; requires fusion to protein interaction partners for complementation-based detection
Needs compatible illumination hardware and optical access. Independent follow-up evidence is still limited. Validation breadth across biological contexts is still narrow. Independent reuse still looks limited, so the evidence base may be fragile. No canonical validation observations are stored yet, so context-specific performance remains under-specified.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
BiFC-PALM revealed nanoscale clustering and diffusion of individual KRas G12D/CRaf RBD complexes on the cell membrane.
BiFC-PALM provided molecular-scale insights into Ras/Raf interaction that would be difficult to obtain with conventional BiFC, fluorescence co-localization, or FRET.
BiFC-PALM enables visualization of protein-protein interactions inside cells with nanometer spatial resolution and single-molecule sensitivity.
The split PAmCherry1 BiFC probe exhibits high specificity and high efficiency at 37°C with virtually no background from spontaneous reconstitution in detecting protein-protein interactions.
background from spontaneous reconstitution virtually no backgroundtemperature 37 °C
Reconstituted split PAmCherry1 maintains fast photoconversion, high contrast ratio, and single-molecule brightness of the parent PAmCherry1, enabling selective PALM localization of protein-protein interactions with about 18 nm spatial precision.
spatial precision 18 nm
PAmCherry1 can function as a BiFC probe when split between residues 159 and 160 into two fragments.
Approval Evidence
1 source3 linked approval claimsfirst-pass slug split-pamcherry1-bifc-probe
We demonstrated that PAmCherry1, a photoactivatable fluorescent protein commonly used for PALM, can be used as a BiFC probe when split between residues 159 and 160 into two fragments.
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performancesupports
The split PAmCherry1 BiFC probe exhibits high specificity and high efficiency at 37°C with virtually no background from spontaneous reconstitution in detecting protein-protein interactions.
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photophysical property retentionsupports
Reconstituted split PAmCherry1 maintains fast photoconversion, high contrast ratio, and single-molecule brightness of the parent PAmCherry1, enabling selective PALM localization of protein-protein interactions with about 18 nm spatial precision.
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probe designsupports
PAmCherry1 can function as a BiFC probe when split between residues 159 and 160 into two fragments.
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Comparisons
Source-stated alternatives
The abstract frames this probe against conventional BiFC probes and against non-complementation approaches such as fluorescence co-localization or FRET.
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The abstract frames this probe against conventional BiFC probes and against non-complementation approaches such as fluorescence co-localization or FRET.
Source-backed strengths
high specificity; high efficiency even at 37°C; virtually no background from spontaneous reconstitution; maintains fast photoconversion, high contrast ratio, and single molecule brightness
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high specificity
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high efficiency even at 37°C
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virtually no background from spontaneous reconstitution
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maintains fast photoconversion, high contrast ratio, and single molecule brightness
Compared with FRET
The abstract frames this probe against conventional BiFC probes and against non-complementation approaches such as fluorescence co-localization or FRET.
Shared frame: source-stated alternative in extracted literature
Strengths here: high specificity; high efficiency even at 37°C; virtually no background from spontaneous reconstitution.
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The abstract frames this probe against conventional BiFC probes and against non-complementation approaches such as fluorescence co-localization or FRET.
Compared with in vivo bimolecular fluorescence complementation
The abstract frames this probe against conventional BiFC probes and against non-complementation approaches such as fluorescence co-localization or FRET.
Shared frame: source-stated alternative in extracted literature
Strengths here: high specificity; high efficiency even at 37°C; virtually no background from spontaneous reconstitution.
Source:
The abstract frames this probe against conventional BiFC probes and against non-complementation approaches such as fluorescence co-localization or FRET.
Ranked Citations
- 1.