Methods and systems for monitoring intracellular binding reactions
AssigneeCaliper Technologies Corp.
InventorsH. Garrett Wada, Javier A. Farinas, Theo T. Nikiforov
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Abstract
Intracellular binding reactions, and particularly DNA/DNA binding protein reactions are detected in situ, using intracellular fluorescence polarization detection. The methods comprise providing a biological cell having at least a first component of a binding reaction disposed therein. The cell is contacted with a second component of the binding reaction whereby the second component is internalized within the biological cell. At least one of the first and second components has a fluorescent label. The amount of binding between the first and second components within the cell is determined by measuring a level of polarized and/or depolarized fluorescence emitted from within the biological cell.
Claims33
A method of detecting intracellular binding interactions, comprising:
- providing a biological cell having at least a first component of a binding reaction disposed therein;
- contacting the cell with a second component of the binding reaction whereby the second component is internalized within the biological cell, the second component comprising a non-protein molecule, at least one of the first and second components having a fluorescent label associated therewith;
- and
- determining an amount of binding between the first and second components within the cell by measuring a level of polarized fluorescence emitted from within the biological cell.
A method of monitoring activation of a cell signaling pathway, comprising:
- providing a cell which comprises a cell signaling pathway, wherein at least one step in the cell signaling pathway comprises an intracellular binding interaction between a nucleic acid and a nucleic acid binding protein;
- contacting the cell with a first nucleic acid whereby the first nucleic acid is internalized within the cell, wherein the first nucleic acid comprises a fluorescent label and is capable of being bound by the nucleic acid binding protein;
- and
- monitoring a level of binding between the nucleic acid binding protein and the first nucleic acid by monitoring a level of depolarized fluorescence emitted from within the cell, the amount of depolarized fluorescence being indicative of a level of activation of the cell signaling pathway.
A method of screening for effectors of cellular function, comprising:
- providing at least a first cell capable of at least one cellular function that is initiated by interaction of a nucleic acid sequence and a nucleic acid binding protein;
- inserting a first nucleic acid having a fluorescent label associated therewith into the cell, wherein the first nucleic is capable of being bound by the nucleic acid binding protein;
- exposing the cell to a test compound;
- determining an amount of binding between the first and second components within the cell by measuring a level of polarized fluorescence emitted from within the biological cell;
- and
- comparing an amount of binding between the first and second components within the cell in the presence of the test compound to an amount of binding between the first and second components within the cell in the absence of the test compound.
Dependencies are inferred from the claim text.