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Methods of screening for ligands of target molecules

  • US 20030059811A1
  • Filed: 06/13/2002
  • Published: 03/27/2003
  • Est. Priority Date: 06/14/2001
  • Status: Abandoned Application
First Claim
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1. A method of screening to identify one or more ligands that bind to a target molecule, comprising the steps:

  • (a) providing a target molecule in solution in one or more wells;

    (b) adding to said one or more wells one or more test compounds to provide one or more test wells comprising a target molecule and one or more test compounds;

    (c) adding to said one or more test wells a first specific binding member that specifically binds the unfolded form of said target molecule, wherein said first specific binding member comprises a FRET donor or a FRET acceptor or can directly or indirectly bind a FRET donor or a FRET acceptor;

    (d) subjecting said one or more test wells to conditions at which at least a portion of said target molecule is denatured;

    (e) adding to said one or more test wells a second specific binding member that can bind said target protein at a site distinct from the binding site of said first specific binding member, wherein;

    when said first specific binding member comprises or can directly or indirectly bind a FRET donor fluorophore, said second specific binding member comprises or can directly or indirectly bind a FRET acceptor, and when said first specific binding member comprises or can directly or indirectly bind a FRET acceptor, said second specific binding member comprises or can directly or indirectly bind a FRET donor;

    (f) measuring fluorescence emission at one or more wavelengths from said test wells;

    (g) making a comparison of fluorescence emission at one or more wavelengths of said one or more test wells with one or more reference values;

    (h) using said comparison in step (g) to determine the extent to which said target molecule occurs in the unfolded state, the folded state, or both in said one or more test wells; and

    (i) using the determination in part (h) to determine whether said one or more test compounds binds said target molecule, thereby identifying one or more ligands of said target molecule.

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