Split-GFP quantification is an innovative approach for real-time protein quantification and localization studies. Our platform utilizes engineered split-GFP systems to provide precise, fluorescence-based measurements of protein concentration and cellular localization.

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Technology Principle

The split-GFP system divides the green fluorescent protein (GFP) into two non-fluorescent fragments that can reconstitute to form a functional, fluorescent protein when brought into proximity.

GFP Fragment 1-10

Large fragment containing β-strands 1-10

  • Stable, non-fluorescent fragment
  • Can be expressed as fusion protein
  • High affinity for complementary fragment

GFP Fragment 11

Small fragment containing β-strand 11

  • Short peptide sequence (16 amino acids)
  • Minimal disruption to target protein
  • Rapid complementation kinetics

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Quantification Methods

Principle: Target protein fused to GFP fragment 1-10, complemented with free fragment 11

Applications:

  • Protein expression monitoring
  • Real-time production tracking
  • Yield optimization
  • Quality control

Advantages:

  • Linear fluorescence response
  • High sensitivity detection
  • Real-time monitoring
  • Minimal protein perturbation

Typical sensitivity: 10 nM to 10 μM protein concentration

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Experimental Design

1

Fragment Selection

Choose appropriate GFP fragments based on experimental requirements.

2

Protein Engineering

Design fusion constructs with optimal fragment placement.

Considerations:

  • Fragment positioning (N-term, C-term, internal)
  • Linker design and length
  • Structural compatibility
  • Functional preservation

Our team provides computational modeling to predict optimal fragment placement for your specific proteins.

3

Expression and Complementation

Express fragments individually or co-express for complementation studies.

Expression systems:

  • Cell-free expression: Rapid screening
  • Bacterial expression: High-yield production
  • Mammalian expression: Native environment
  • Yeast expression: Eukaryotic processing

Complementation typically occurs within minutes to hours depending on fragment concentrations and binding affinity.

4

Fluorescence Detection

Monitor fluorescence using appropriate detection systems.

Detection methods:

  • Fluorescence microscopy: Single-cell analysis
  • Flow cytometry: Population analysis
  • Plate readers: High-throughput screening
  • Spectrofluorometry: Precise quantification

Detection sensitivity allows measurement of nanomolar protein concentrations in real-time.

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Applications

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Protein Expression Monitoring

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Binding Affinity Measurements

Equilibrium Binding

Steady-state measurements for KD determination

  • Saturation binding curves
  • Competitive binding assays
  • Cooperative binding analysis
  • Multi-site binding studies

Kinetic Analysis

Real-time binding kinetics for rate constants

  • Association rate (kon) measurement
  • Dissociation rate (koff) measurement
  • Binding mechanism analysis
  • Kinetic selectivity studies

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Cellular Localization

Quantitative localization analysis:

  • Organelle-specific localization
  • Membrane vs. cytoplasmic distribution
  • Nuclear vs. cytoplasmic ratios
  • Localization coefficient calculation

Applications:

  • Trafficking studies
  • Organelle targeting
  • Localization signal analysis
  • Disease-related mislocalization

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Advantages Over Traditional Methods

Sensitivity

Higher sensitivity than traditional methods

  • Single-molecule detection capability
  • Low background fluorescence
  • High signal-to-noise ratio
  • Quantitative measurements

Real-Time Monitoring

Live-cell compatible measurements

  • Continuous monitoring
  • Dynamic process tracking
  • Temporal resolution
  • Non-invasive detection

Minimal Perturbation

Small tag size reduces protein disruption

  • Fragment 11 only 16 amino acids
  • Minimal structural impact
  • Preserved protein function
  • Native-like behavior

Versatility

Multiple experimental formats

  • In vitro and in vivo compatible
  • Various expression systems
  • Different detection methods
  • Flexible experimental design

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Data Analysis and Quantification

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Fluorescence Quantification

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Kinetic Analysis

Binding kinetics:

  • Association rate constants
  • Dissociation rate constants
  • Equilibrium dissociation constants
  • Cooperativity parameters

Expression kinetics:

  • Production rates
  • Degradation rates
  • Steady-state levels
  • Time-to-maximum expression

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Integration with Other Technologies

Split-GFP quantification complements other analytical methods:

  • Binding assays: Orthogonal binding measurements
  • Structural studies: Functional validation
  • Purification: Real-time monitoring
  • Quality control: Rapid assessment

GFP complementation is irreversible under normal conditions. Consider this when designing experiments requiring reversible interactions.

Fragment placement optimization is crucial for maintaining protein function. Our team provides computational and experimental optimization services.