BRET Procedures

BRET measurements in vivo: Incubate (co-)transformed tissue with 1-5uM coelenterazine for 0-15 minutes and measure luminescence for 10 second periods through 460nm (50nm width; i.e.+/-25nm) and 535nm (40nm width) filters. We take four pairs of readings each through blue and yellow filters (B-Y-B-Y-B-Y-B-Y) and average the data before calculating the BRET ratio. Pay attention to ‘drifts’ of the readings over this time – luminescence should be pretty stable.
How to work with coelenterazine (MW 423.47)

Sources: Biotium Inc. (3423 Investment Blvd. Suite 8, Hayward, CA 94545, USA. Phone: 510-265-1027). Prolume (www. prolume.com, Pinetop, AZ). Biosynth AG (will only sell large amounts at this time).
Stock at 250uM (250x): dissolve 1mg coelenterazine in 9.44ml 100% ethanol.Prepare 400ul aliquots and 40ul (most useful) and 20ul aliquots in 1.5ml tubes. Dry down with speed vac and gas with N2 (nitrogen), seal, and store at -80C in a black box. - Reconstitute coelenterazine by adding 40ul 100% ethanol to a '40ul aliquot'. This is a 250x stock. Our final concentration of coelenterazine is 1uM, but others use higher concentrations (3-10uM). I find it useful to add another 960ul water to make 1ml of a 10x stock. Keep tube wrapped in foil and on ice throughout the experiment. If you have leftover coelenterazine at the end of the day you can store it at –70oC but its activity will dwindle.
DeepBlueC

Perkin-Elmer is marketing the BRET2 system, in which the spectral shift between LUC and FP is increased, thus providing more sensitive BRET detection. BRET2 is based on a specially formulated substrate for RLUC, which emits purple photons (bisdeoxycoelenterazine). This is combined with a GFP-variant whose excitation is tailored to the ‘DeepBlueC’ substrate. We have not been successful with using DeepBlueC for RLUC in plants, either onion cells in vivo or Arabidopsis protein extract. Try it in mammalian cells.
Detailed BRET assay using the Turnerdesigns TD-20/20 with the ‘dual color accessory':

This procedure is for in vivo expression of luciferase and YFP fusion proteins in onion epidermal cells (biolistic transformation) or in Arabidopsis thaliana seedlings (biolistic transformation or transgenic Arabidopsis seedlings).
1. DNA containing the BRET-expression cassettes is transformed into the appropriate cells.
2. The tissue is placed into a 1.5 ml clear eppendorf tube or 12 X 50 round bottom polypropylene tube (available from Turner Designs) containing 0.5 ml water.
3. 50 microliter of 10 micromolar coelenterazine substrate is added for a final concentration of 1micromolar.
4. Mix by tapping, then take luminescence readings within 30 seconds using the TD-20/20. The readings obtained are in Relative Luminescence Units (RLU). The luminescence is measured after a 2 seconds delay and over a 10 seconds integration period after pressing <GO> on the instrument keypad.
5. It is recommended to run a time course by taking readings after 1min, 2 min, 5 min, 15 min, 30 min, 45 min and 1 hour. In our experience in most cases the luminescence increased up to 30 minutes and then decreased.
6. Replicate readings can be taken by pressing SETUP then <1> then <3> on the instrument keypad. If the instrument is connected to a computer then the readings are recorded on to an Excel spreadsheet as r1, r2, r3 if three replicates are chosen. At the end of the third replicate, the average, %CV and standard deviation is also displayed.
In vitro BRET measurements: Luciferase levels are usually manyfold higher in vitro than in vivo, but the half life of LUC activity in an extract is on the order of seconds. Much of the luminescence may be emitted as a flash immediately after substrate injection. For comparison, in vivo luminescence can be characterized as ‘glow’ luminescence.
Filters for BRET

A variety of filters can be used to measure BRET. Filters for items (2) and (3) below are standard 26mm diameter microscope filters available from Chroma or Omega.
1. The Turnerdesigns light switch module (off-the-shelf accessory for the TD20/20, inquire about specific transmission characteristics)
  
  Blue: 333nm-463nm with >90% transmission from 370-410nm
  Yellow: 520nm longpass with >80% transmission from 550nm up
  These are Wratten gel filters.
  Note: Consider using a filter with higher transmission in the 460-490nm range.
2. BMG Labtech PolarStar, built in filters; measures blue and yellow simultaneously.
3. Packard Fusion for standard BRET (built-in):            for BRET2:
  Blue: 485/20                                                         410 nm
  Yellow: 540/25 or 530/25                                       515 nm(DeepBlueC).
4. Wallac plate reader:
  
  Blue: 460nm /50nm width (i.e 460+/-25nm; Chroma)
  Yellow: 535/40 (Chroma). (can also try 585/40 or 550 longpass).
Alternative extraction buffers for BRET

A
50mM KCl,
50mM NaCl,
2.5mM MgCl2,
2 mM EDTA,
5mM DTT,
0.2%NP-40,
10ug/ml PMSF (see note below)
2ug/ml aprotinin,
2ug/ml leupeptin,
20mM HEPES pH8;
- for E. coli add 10mg/ml (!) lysozyme

B
500mM NaCl
100mM potassium phosphate pH7.4
1mM EDTA
add 0.02% BSA
- optional:1mM DTT

Buffer A was developed by Yao Xu and Carl Johnson. Note that RLUC may be inhibited by PMSF. Buffer B is a high salt buffer used by Mayerhofer and Szalay, adapted from Matthews and Cormier (1977), which stimulates the luciferase activity, but luciferase is less stable over time than with buffer A. In general, LUC activities drop with a half-life of a few minutes (A) or a few seconds (B) upon addition of substrate.

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