Kinase and ubiquination assays
View Patent ↗Compositions, including antibodies, polypeptides, and organic molecules, kits, and methods for probing molecular interactions (e.g., deubiquination, ubiquination and kinase activity), e.g., using resonance energy transfer (RET) are provided.
1. A method for detecting at least one substrate for at least one ubiquitination or ubiquitination-like enzyme, the method comprising:
a) contacting i) the at least one ubiquitination or ubiquitination-like enzyme with ii) polypeptides immobilized on a support, wherein the polypeptides are associated with a first detectable moiety, and iii) at least one ubiquiton comprising a second detectable moiety, wherein the first moiety and the second moiety comprise a resonance energy transfer (RET) pair,
b) incubating (a) under conditions to allow for ubiquitination or ubiquitination-like activity,
c) exposing the support to at least one wavelength of light, and
d) detecting at least one substrate for at least one ubiquitination or ubiquitination-like enzyme by detecting resonance energy transfer from the support.
2. A method for identifying deubiquitinating activity of a sample, the method comprising:
a) contacting i) the sample with ii) at least one or a plurality of polypeptides immobilized on a substrate, wherein the at least one or plurality of polypeptides is associated with a first detectable moiety and wherein the at least one or plurality of polypeptides comprise a ubiquitin or ubiquitin-like protein associated with a second detectable moiety, wherein the first moiety and the second moiety comprise a RET pair and wherein the deubiquitinating activity causes the dissociation of the detectable moiety from the substrate,
b) incubating (a) under conditions suitable for de-ubiquitination activity,
c) exposing the substrate to at least one wavelength of light, and
d) detecting fluorescence emission from the substrate;
wherein a decrease in RET between the first and second moiety indicates deubiquitinating activity in the sample.
3. The method of claim 1 , wherein the first detectable moiety is a fluorescent protein.
4. The method of claim 1 , wherein the polypeptides are associated with an antibody and the antibody comprises the first detectable moiety.
5. The method of claim 1 , wherein the at least one ubiquiton is directly labeled with the second detectable moiety.
6. The method of claim 1 , wherein the first detectable moiety comprises a luminescent metal complex and the second detectable moiety comprises a fluorescent acceptor moiety.
7. The method of claim 2 , wherein the at least one or plurality of polypeptides is a fluorescent protein.
8. The method of claim 7 , wherein the second detectable moiety comprises a luminescent metal complex.
9. The method of claim 2 , wherein the at least one or plurality of polypeptides and the ubiquitin or ubiquitin-like protein are fused as part of a fusion protein.
10. The method of claim 2 , wherein the second detectable moiety is covalently associated with the ubiquitin or ubiquitin-like protein.
11. The method of claim 2 , wherein the first detectable moiety comprises a luminescent metal complex and the second detectable moiety comprises a fluorescent acceptor moiety or the first detectable moiety comprises a fluorescent acceptor moiety and the second detectable moiety comprises a luminescent metal complex.
12. A method for detecting at least one substrate for at least one ubiquitination or ubiquitination-like enzyme, the method comprising:
a) contacting i) the at least one ubiquitination or ubiquitination-like enzyme with ii) polypeptides immobilized on a support, iii) at least one ubiquiton comprising a first detectable moiety, and iv) at least one ubiquiton comprising a second detectable moiety, wherein the first moiety and the second moiety comprise a resonance energy transfer (RET) pair,
b) incubating (a) under conditions to allow for ubiquitination or ubiquitination-like activity,
c) exposing the support to at least one wavelength of light, and
d) detecting at least one substrate for at least one ubiquitination or ubiquitination-like enzyme by detecting resonance energy transfer from the support.
13. The method of claim 12 , wherein the first detectable moiety is directly associated with the at least one ubiquiton.
14. The method of claim 13 , wherein the second detectable moiety is indirectly associated with the at least one ubiquiton.
15. The method of claim 12 , wherein the first detectable moiety comprises a luminescent metal complex and the second detectable moiety comprises a fluorescent acceptor.
16. A method for detecting at least one substrate for at least one ubiquitination or ubiquitination-like enzyme, the method comprising:
a) contacting i) the at least one ubiquitination or ubiquitination-like enzyme with ii) a positionally addressable array comprising polypeptides immobilized on a support, iii) at least one ubiquiton comprising a first detectable moiety, and iv) at least one ubiquiton comprising a second detectable moiety, wherein the first moiety and the second moiety comprise a resonance energy transfer (RET) pair, wherein the detectable moiety comprises a fluorescent or luminescent label,
b) incubating (a) under conditions to allow for ubiquitination or ubiquitination-like activity,
c) exposing the array to at least one wavelength of light, and
d) detecting at least one substrate for at least one ubiquitination or ubiquitination-like enzyme by detecting resonance energy transfer from the support.
17. The method of claim 16 , wherein the detectable moiety is indirectly associated with the at least one ubiquiton.