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Why tethered-ligand technology?

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Why tethered-ligand technology? It is easy to bind targets to microarrays, but in order to detect interactions, the fluorescence of a spot must either increase or ... – PowerPoint PPT presentation

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Title: Why tethered-ligand technology?


1
Why tethered-ligand technology?
  • It is easy to bind targets to microarrays, but in
    order to detect interactions, the fluorescence of
    a spot must either increase or decrease
  • Detection is easy for genomic microarrays
    standard DNA/RNA enzymatic methods amplify and
    fluorescently label nucleic acid samples
  • Detection is very difficult for proteomic
    microarrays no equivalent enzymatic
    amplification and duplication methods
  • Direct chemical labeling methods are
    unsatisfactory

2
Labeling genomic vs proteomic
3
Chemical labeling can change protein-target
interactions
4
How to detect binding of unlabeled proteins and
ligands?
  • Want to simply take a cell extract or diagnostic
    sample, dump it on a microarray, and go
  • The microarray itself must contain the detection
    means in an inactive state

5
Tethered-ligand detection techniques
  • Relies on fluorescence resonance transfer (FRET)
    technology
  • An FRET labeled receptor-ligand reagent is
    prebound to the microarray
  • Analytes are detected as they perturb the bound
    receptor-ligand pair

6
Miyatas Antigen-Ab hydrogels
Nature 399, 766 - 769 (1999)
7
What is fluorescence resonance transfer (FRET)
technology?
Ro
  • Energy transfer between two dye molecules
  • Ro distance is typically between 40 to 90
    angstroms
  • Commonly used technique

Dye 1 Dye 2
8
Tethered-ligand chemistry
Here, a labeled ligand is displaced from a
receptor by an unlabeled test sample ligand
9
Enzymatic detection
-P
Here, a kinase phosphorylates a group, abolishing
target binding, and creating a fluorescent
signal.
10
General purpose reagent platform?
  • Cover microarray surface with FRET coupled
    Avidin and 2nd antibody
  • In selected locations, bind biotinated ligand
    1st antibody complex
  • Addition of unlabeled ligand will abolish Avidin
    2nd antibody FRET

11
Tetered-ligand patent application 20030108972 A1
  • Concept of tethered-ligand methods for all
    microarray applications
  • All detection methodologies and enzymatic types
    claimed
  • Specific applications for proteases, kinases,
    sepsis, apoptosis, angiogenesis
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