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BRET Advice

Please contact Albrecht von Arnim with requests for vectors, procedures, or advice. Use procedures at your own risk!

* Important. Be meticulous in setting up controls with untransformed tissue (autoluminescence) and with RLUC alone (no YFP acceptor). Differences in pigment content can easily suggest BRET when in fact all you are measuring is chlorophyll content. Detergents and proteins (BSA...) may cause autoluminescence of coelenterazine.

  1. The distance requirement: Like other Förster transfers, the BRET efficiency is extremely sensitive to distance between the interaction partners. Therefore, your chances for success (in detecting BRET) increase if you prepare both N- and C-terminal fusions to RLUC and YFP (four constructs per protein). This gives you four possible combinations if you look for homodimerization and eight combinations when looking for heterodimerization! Your goal should be to achieve immediate juxtaposition of the RLUC and YFP tags (~5nm distance).

    With small proteins (<10kDa), it may not matter which way you fuse the BRET tags. With medium sized proteins (50kDa) it will matter.

  2. Cell types: We have expressed BRET tagged proteins in onion epidermal cells and Arabidopsis seedlings, in stably transformed tobacco BY2 cells, in stably transformed Arabidopsis, and in Arabidopsis leaves infiltrated with Agrobacterium. Most of our BRET data come from onion cells. We routinely check for RLUC and YFP expression using particle bombardment of onion epidermal cells before co-expressing the proteins to look for BRET.

  3. The substrate: RLUC uses the substrate coelenterazine. We routinely work with standard underivatized coelenterazine at 1 micromolar, but up to 10 micromolar can be used (see instructions below). See note below on DeepBlueC.

  4. The luminometer: We use the Turnerdesigns TD-20/20 with a custom-made 'LightSwitch module' also known as the Dual Color Accessory. See detailed instructions below. In principle, any luminometer in which you can filter the signal through blue and yellow filters will work (see notes below), but in practice, several models of microplate luminometers have insufficient sensitivity, except for the strongest LUC signals. It is possible to operate the TD-20/20 with standard ‘microscope’ filters (26mm diameter), instead of the dual-color accessory, but the manual swapping of the filters between measurements will generate variability. Also beware of filters that 'glow' (delayed fluorescence).

Expected results

With our setup in onion cells, RLUC gives a Y:B ratio of 0.6 and RLUC-YFP fusion (good BRET) gives about 1.2-1.4. A Y:B ratio of 0.8 or lower may be statistically significant.

Medium for BRET

For onion tissue we simply use water. For Arabidopsis we use 1/2xMS salts pH 5.7 and 0.01% Silwet. 1% sucrose may be added. RLUC activity will be higher if tissue is vacuum treated, but survival of plants may be poor.