Complex BRET technique for measuring biological interactions
Provided are methods for detecting protein interactions in a sample, the methods comprising: (a) detecting two or more polypeptides that when associated emit a first detectable signal in a first light emission spectrum; (b) contacting the two or more polypeptides with a third polypeptide conjugated to a dipole acceptor moiety that has a second light emission spectrum when excited within a light excitation spectrum, wherein the light excitation spectrum overlaps with the first light emission spectrum; and (c) detecting a second detectable signal emitted in the second light emission spectrum by the dipole acceptor moiety. Also provided are bioluminescent complexes comprising: (a) a first polypeptide conjugated to a dipole acceptor moiety, wherein the emits a first detectable signal in a first light emission spectrum.
1. A method for detecting protein interactions in a sample, the method comprising:
(a) detecting a first polypeptide and a second polypeptide that when associated emit a first detectable signal in a first light emission spectrum, wherein the first polypeptide is conjugated to a first non-luminescent element and the second polypeptide is conjugated to a second non-luminescent element, wherein the first and second non-luminescent elements form a complex that emits the first detectable signal;
(b) contacting the first polypeptide and the second polypeptide with a third polypeptide conjugated to a dipole acceptor moiety that has a second light emission spectrum when excited within a light excitation spectrum, wherein the light excitation spectrum overlaps with the first light emission spectrum, wherein the contacting places the first polypeptide and the second polypeptide in a proximity sufficient to allow resonance energy transfer from the first and the second polypeptides to the dipole acceptor moiety, thereby exciting the dipole acceptor moiety to emit a second detectable signal;
(c) detecting the second detectable signal emitted in the second light emission spectrum by the dipole acceptor moiety and
(d) determining (i) an increase in a ratio of the second detectable signal:the first detectable signal after the contact between the first polypeptide, the second polypeptide, and the third polypeptide, as compared to a ratio of the second detectable signal: the first detectable signal before the contact between the first polypeptide, the second polypeptide, and the third polypeptide, or (ii) an increase in the second detectable signal following association between the first polypeptide, the second polypeptide and the third polypeptide,
wherein the increase in (i) or (ii) indicates that the first polypeptide, the second polypeptide and the third polypeptide interact to form a complex.
2. The method of claim 1 , wherein the dipole acceptor moiety comprises a fluorescent moiety, a dye, an arsenical protein label, a dye-protein conjugate pair, or a quantum dot.
3. The method of claim 1 , wherein the dipole acceptor moiety comprises a fluorescent moiety.
4. The method of claim 3 , wherein the second light emission spectrum is within a range of from about 445 nm to about 700 nm.
5. The method of claim 1 , wherein the first non-luminescent element and the second non-luminescent element each independently comprise a portion of a luciferase enzyme.
6. The method of claim 1 , wherein the sample comprises a cell, and the contacting is performed by molecular interactions in the cell.