BRET Principle Efficiency and Instrumentation

The efficiency of BRET is dependent upon factors such as spectral properties, relative distance, and orientation of donor and acceptor molecules [6, 7]. BRET normally occurs when the distance between the donor and the acceptor is within 100 A, and the efficiency of BRET is inversely related to the distance to the sixth power. The rate of energy transfer is given by Förster's equation, fcr = (1/Td) (-R0/-R)6, where fcj. is the rate of energy transfer, Td is the fluorescence lifetime of the donor in the absence of the acceptor, R0 is the Förster critical radius at which 50% of the excitation energy is transferred to the acceptor, and R is the distance between the centers of the donor and acceptor chromophores. Thus, the relative location between the donor molecule and the acceptor is critical for an efficient energy transfer.

Fig. 6.2 Emission spectrum of Rluc (solid line) and excitation and emission spectrum of YFP (dotted line). The spectral overlap between the emission of RLuc and the excitation of YFP is evident from the spectra. Adapted from Ref. [25]. Reprinted with permission from Elsevier Science.

In addition to the distance, when designing fusion proteins for BRET assays, consideration should be given to the relative orientation of the donor and acceptor molecules. There should be sufficient flexibility between the donor and the acceptor in a fusion protein. It is necessary that the spectrum of donor emission and acceptor excitation overlap significantly in order to achieve a BRET of high efficiency (Fig. 6.2). Spectral resolution is lost if the donor and acceptor emission spectrum overlap, resulting in a low signal-to-noise ratio.

Luminescence and fluorescence measurements are performed in many labo-rat ories routinely. Therefore, there is interest in developing instruments that allow sensitive and easy detection of light from emission-based methods. A large number of instruments capable of high-throughput measurements have been developed in recent years. These instruments are capable of working with traditional sample volumes, such as 96-well plate readers, as well as with low sample volumes, which are based on 384-well and 1536-well plates [6]. Some of these instruments have the ability to perform measurement in multiple modes such as absorbance, fluorescence, luminescence, fluorescence polarization, and anisotropy [6]. Typically, BRET measurements are performed by using either a microtiter plate reader or a scanning spectroscopy-type of instrument. In microtiter plate readers, the light is collected using fiber optics and a filter wheel that allows selection of emission from the fluorophore, which is kept in the path of the light. A highly sensitive PMT is used as a detector. Careful consideration should be given while selecting the bandpass filter, such that the signal corresponding to the donor emission is not collected. To overcome this drawback, some companies have made available BRET-optimized filters that allow longer bandpass for excitation and smaller bandpass for fluorophore emission measurement [6].

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