P27 ubiquitination assay and methods of use
First Claim
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1. A method of determining the amount of ubiquitination of a protein, comprising:
- a. contacting said protein with a plurality of polypeptides, said plurality of polypeptides together capable of ubiquitinating said protein, to form ubiquitinated protein;
b. capturing said ubiquitinated protein on a surface; and
c. determining an amount of ubiquitin present in said protein captured on said surface, wherein the amount of ubiquitin present in said protein captured on said surface is correlated with the amount of ubiquitination of said protein.
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Abstract
This invention relates to easy and reliable assays for the ubiquitination of p27. Because the assay can be performed as a plate capture assay, or as a homogenous time-resolved fluorescence resonance energy transfer assay, with defined, easily replicatable reaction conditions, it is particularly useful for high-throughput screening of, for example, a prospective anticancer agent. The invention provides methods to determine the amount of ubiquitination of p27, or of any protein, and use of the method to identify compounds that modulate the ubiquitination of p27, or any protein.
14 Citations
49 Claims
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1. A method of determining the amount of ubiquitination of a protein, comprising:
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a. contacting said protein with a plurality of polypeptides, said plurality of polypeptides together capable of ubiquitinating said protein, to form ubiquitinated protein;
b. capturing said ubiquitinated protein on a surface; and
c. determining an amount of ubiquitin present in said protein captured on said surface, wherein the amount of ubiquitin present in said protein captured on said surface is correlated with the amount of ubiquitination of said protein. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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- 11. A method of identifying a compound that modulates ubiquitination of p27, comprising determining the amount of ubiquitinated p27 formed by combining of isolated phosphorylated p27, E1, E2, E3, Cks1 and ubiquitin in the presence of said compound and in the absence of said compound, wherein, if the amount of ubiquitinated p27 formed in the presence of said compound differs from the amount of ubiquitinated p27 formed in the absence of said compound, said compound is identified as a compound that modulates the ubiquitination of p27.
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21. A method of determining the amount of ubiquitin in a ubiquitinated protein, comprising:
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a. labeling said protein with a first label;
b. labeling said ubiquitin with a second label;
c. determining a ratio of said second label to said first label, wherein a higher ratio indicates a greater amount of ubiquitination of said protein. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
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34. A method of identifying an anticancer agent, comprising determining the amount of ubiquitinated p27 formed by combining isolated p27, E1, E2, E3, Cks 1, Cyclin E, Cdk2 and ubiquitin in the presence of said compound and in the absence of said compound;
- wherein, if the amount of ubiquitinated p27 formed in the presence of said compound differs from the amount of ubiquitinated p27 formed in the absence of said compound, said compound is identified as an anticancer agent.
- View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42, 43)
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44. A kit for p27 ubiquitination assay comprising p27;
- Cdk2/Cyclin E; and
a plurality of polypeptides, said plurality of polypeptides together capable of ubiquitinating p27, recombinant cells expressing the polypeptides, or recombinant vectors harboring sequences which encode the polypeptides. - View Dependent Claims (45, 46, 47, 48, 49)
- Cdk2/Cyclin E; and
Specification