Methods and kits for assays utilizing fluorescence polarization

A method of determining by fluorescence polarization if a compound binds to a binding partner, said method comprising the steps of:

• a. providing a solution comprising said compound labeled with at least two different fluorophores, wherein:
• i. each fluorophore has a maximal excitation peak that is at least 30 nanometers different from the maximal excitation peak of another fluorophore on said compound and a maximal emission peak that is at least 30 nanometers different from the maximal emission peak of another fluorophore on said compound, and
• ii. there is less than 50% fluorescence resonance energy transfer between any two of the fluorophores present in the labeled compound;
• and
• b. comparing the fluorescent polarization values of said solution in the absence and the presence of said binding partner at multiple wavelengths that correspond to the wavelengths at which each of said fluorophores present on said compound optimally absorbs and emits.

A method of determining by fluorescence polarization if a population of compounds is incorporated into a larger molecule, said method comprising the steps of:

• a. providing a first mixture comprising at least two subpopulations of compounds, wherein:
• i. each subpopulation is characterized by a single compound labeled with a fluorophore;
• ii. the fluorophore in any subpopulation has a maximal excitation peak that is at least 30 nanometers different from the maximal excitation peak of the fluorophore in another subpopulation and a maximal emission peak that is at least 30 nanometers different from the maximal emission peak of the fluorophore in another subpopulation;
• and
• iii. the labeled compound in each of the subpopulations is capable of being incorporated into a larger molecule through the action of an enzyme or a catalyst;
• and
• iv. the labeled compound in each of the subpopulations has a rate of incorporation into a larger molecule through the action of said enzyme or said catalyst that is less than 50-fold different from the rate of incorporation into a larger molecule by said enzyme or said catalyst of the labeled compound in any other of the subpopulations;
• b. measuring the fluorescence polarization values of said first mixture at multiple wavelengths that correspond to the wavelengths at which each of said fluorophores present in said first mixture optimally absorbs and emits;
• c. adding to said first mixture a solution comprising said enzyme or said catalyst capable of incorporating said compounds into a larger molecule to create a second mixture, wherein said second mixture is incubated for a period of time and provides conditions that, in the absence of an inhibitor of said enzyme or said catalyst, allow said compounds to be incorporated into a larger molecule through the action of said enzyme or said catalyst;
• d. measuring the fluorescence polarization values of said second mixture at multiple wavelengths that correspond to the wavelengths at which each of said fluorophores present in said mixture optimally absorbs and emits;
• e. comparing the fluorescence polarization measurements taken at steps b and d to determine if said population of compounds have been incorporated into a larger molecule.

A method of determining by fluorescence polarization if a compound is incorporated into a larger molecule, said method comprising the steps of:

• a. providing a first solution comprising a compound labeled with at least two different fluorophores, wherein:
• i. each fluorophore has a maximal excitation peak that is at least 30 nanometers different from the maximal excitation peak of another fluorophore on said compound and a maximal emission peak that is at least 30 nanometers different from the maximal emission peak of another fluorophore on said compound;
• ii. there is less than 50% fluorescence resonance energy transfer between any two of the fluorophores present in the labeled compound;
• and
• iii. said compound is capable of being incorporated into a larger molecule through the action of an enzyme or a catalyst;
• b. measuring the fluorescence polarization values of said first solution at multiple wavelengths that correspond to the wavelengths at which each of said fluorophores present on said compound optimally absorbs and emits;
• c. adding to said first solution said a solution comprising said enzyme or said catalyst capable of incorporating said compounds into a larger molecule to create a second solution, wherein said second mixture is incubated for a period of time and provides conditions that, in the absence of an inhibitor of said enzyme or said catalyst, allow said compound to be incorporated into a larger molecule through the action of said enzyme or said catalyst;
• d. measuring the fluorescence polarization values of said second solution at multiple wavelengths that correspond to the wavelengths at which each of said fluorophores present in said mixture optimally absorbs and emits;
• and
• e. comparing the fluorescence polarization measurements taken at steps b and d to determine if said compound has been incorporated into a larger molecule.

A method of determining by fluorescence polarization if a population of compounds is degraded into smaller molecules, said method comprising the steps of:

• a. providing a first mixture comprising at least two subpopulations of compounds, wherein:
• i. each subpopulation is characterized by a single compound labeled with a fluorophore;
• ii. the fluorophore in any subpopulation has a maximal excitation peak that is at least 30 nanometers different from the maximal excitation peak of the fluorophore in another subpopulation and a maximal emission peak that is at least 30 nanometers different from the maximal emission peak of the fluorophore in another subpopulation;
• iii. the labeled compound in each of the subpopulations is capable of being degraded into smaller molecules through the action of an enzyme;
• and
• iv. the labeled compound in each of the subpopulations has a rate of degradation through the action of said enzyme that is less than 50-fold different from the rate of degradation by said enzyme of the labeled compound in any other of the subpopulations;
• b. measuring the fluorescence polarization values of said first mixture at multiple wavelengths that correspond to the wavelengths at which each of said fluorophores present in said first mixture optimally absorbs and emits;
• c. adding to said first mixture a solution comprising said enzyme capable of degrading said compounds into smaller molecules to create a second mixture, wherein said second mixture is incubated for a period of time and provides conditions that, in the absence of an inhibitor of said enzyme, allow said compounds to be degraded into smaller molecules through the action of said enzyme;
• d. measuring the fluorescence polarization values of said second mixture at multiple wavelengths that correspond to the wavelengths at which each of said fluorophores present in said mixture optimally absorbs and emits;
• and
• e. comparing the fluorescence polarization measurements taken at steps b and d to determine if said population of compounds have been degraded into smaller molecules.

A method of determining by fluorescence polarization if a compound is degraded into smaller molecules, said method comprising the steps of:

• a. providing a first solution comprising a compound labeled with at least two different fluorophores, wherein:
• i. each fluorophore has a maximal excitation peak that is at least 30 nanometers different from the maximal excitation peak of another fluorophore on said compound and a maximal emission peak that is at least 30 nanometers different from the maximal emission peak of another fluorophore on said compound;
• ii. there is less than 50% fluorescence resonance energy transfer between any two of the fluorophores present in the labeled compound;
• and
• iii. said compound is capable of being degraded into smaller molecules through the action of an enzyme;
• b. measuring the fluorescence polarization values of said first solution at multiple wavelengths that correspond to the wavelengths at which each of said fluorophores present on said compound optimally absorbs and emits;
• c. adding to said first solution a solution comprising said enzyme capable of degrading said compound into smaller molecules to create a second solution, wherein said second mixture is incubated for a period of time and provides conditions that, in the absence of an inhibitor of said enzyme, allow said compound to be degraded into smaller molecules through the action of said enzyme;
• d. measuring the fluorescence polarization values of said second solution at multiple wavelengths that correspond to the wavelengths at which each of said fluorophores present in said mixture optimally absorbs and emits;
• and
• e. comparing the fluorescence polarization measurements taken at steps b and d to determine if said compound has been degraded into smaller molecules.

A kit comprising:

• a. a first subpopulation of a compound labeled with a first fluorophore;
• d. a second subpopulation of a compound labeled with a second fluorophore that has a maximal excitation peak that is at least 30 nanometers different from the maximal excitation peak of the fluorophore in said first subpopulation and a maximal emission peak that is at least 30 nanometers different from the maximal emission peak of the fluorophore in said first subpopulation, wherein said compound in said second subpopulation has:
• i. a dissociation constant for binding to a binding partner that is less than 50-fold different from the dissociation constant for the binding to said binding partner by said compound in said first subpopulation, or
• ii. a rate of incorporation into a larger molecule by an enzyme or a catalyst that is less than 50-fold different from the rate of incorporation into a larger molecule by said enzyme or said catalyst of said compound in said first subpopulation, or
• i. a rate of degradation by an enzyme that is less than 50-fold different from the rate of degradation by said enzyme of said compound in said first subpopulation;
• and
• b. instructions for using said kit which instruct the user to perform fluorescence polarization measurements on a mixture comprising said first and said second subpopulations of compound at multiple wavelengths corresponding to the wavelengths at which each of said first and second fluorophores emits.

A kit comprising:

• a. a compound labeled with at least two fluorophores, wherein:
• i. each fluorophore has a maximal excitation peak that is at least 30 nanometers different from the maximal excitation peak of another fluorophore on said compound and a maximal emission peak that is at least 30 nanometers different from the maximal emission peak of another fluorophore on said compound, and
• ii. there is less than 50% fluorescence resonance energy transfer between any two of the fluorophores present in the labeled compound;
• and
• b. instructions for using said kit which instruct the user to perform fluorescence polarization measurements on a solution comprising said compound at multiple wavelengths corresponding to the wavelengths at which each of said fluorophores emits.

A kit comprising:

• a. a compound;
• b. a first fluorophore;
• c. a second fluorophore that has a maximal emission peak that is at least 30 nanometers different from the maximal absorbance peak of the first fluorophore, wherein there is less than 50% energy transfer between said first and said second fluorophores when said compound is labeled with said fluorophores;
• and
• d. instructions for using said kit which instruct the user to:
• i. label said compound with said first and said second fluorophore;
• ii. perform fluorescence polarization measurements on a solution comprising said labeled compound at multiple wavelengths corresponding to the wavelengths at which each of said fluorophores emits.