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| BRET Protocols Introduce |
| 来源：科端生物 点击数： |
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BRET-Protocols
Here is some preliminary advice for BRET-Experiments. Use procedures at your own risk!
1. To currently have two sets of BRET vectors. pBS-35S-RLUC and pBS-35S-YFP allow you to make fusions to either the N-terminus or C-terminus of RLUC /YFP. A second set of two Renilla luciferase (RLUC) vectors and two YFP vectors features short stretches of alanines as spacers between the fusion partner. The plasmid sequences are available in Genbank: AY189980 to AY189981 (basic N and C-terminal fusions to RLUC and YFP) and AY18992 to AY18995 (fusions with alanine-linkers). 2. As is typical with fusions to fluorescent proteins, some of fusions to Renilla luciferase have high luciferase activity and others don't. Small proteins (<30kDa) are more likely to retain high activitiy than large ones (>60kDa). Some small proteins retain luciferase levels almost as high as the unfused luciferase. 3. Because BRET is very sensitive to 'distance' between the interaction partners your chances for success (in detecting BRET) are increased 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! With small proteins (<10kDa), it may not matter which way use fuse the BRET tags. With medium sized proteins (50kDa) it will matter. In one example, one out of three combinations tested gave evidence of homodimerization. 4. We routinely check for RLUC and YFP expression using particle bombardment of onion epidermal cells before coexpressing the proteins to look for BRET. A ‘good’ RLUC fusion will give activity 100-fold above the background noise of the instrument (and ‘bad’ fusions are almost inactive). 5. We have expressed BRET tagged proteins in onion epidermal cells, in stably transformed BY2 cells, in stably transformed Arabidopsis, and in Arabidopsis leaves infiltrated with Agrobacterium. Most of our BRET data come from onion cells. 6. RLUC uses the substrate coelenterazine. We routinely work with it at 1 micromolar, but up to 10 micromolar can be used (see instructions below). See note below on DeepBlueC. 7. Luminometer: 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, microplate luminometers have insufficient sensitivity, except for the strongest LUC signals (inquire). Also beware of filters that 'glow' (delayed fluorescence). 8. BRET measurements in vivo: Incubate (co-)transformed tissue with 1-5uM coelenterazine for 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. In vitro BRET measurements: See the notes below. Luciferase levels are usually manyfold higher in vitro than in vivo, but the halflife of LUC activity in an extract is on the order of a few minutes. RLUC is said to be stimulated by high salt (500mM NaCl). 9. Medium for BRET: For onion tissue we simply use water. For Arabidopsis we use 1/2xMS salts pH5.7 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. 10. Expected results. With our setup in onion cells, RLUC gives a Y:B ratio of 0.6 and good BRET (RLUC-YFP fusion) gives about 1.2-1.4. A Y:B ratio of 0.8 may be statistically significant. 11. 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.
How to work with coelenterazine (MW 423.47) 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 some people 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. Luciferase assay using Tube Luminometer with the ‘dual color Filter': This procedure is for in vivo expression of luciferase in onion epidermal cells (biolistic transformation) or in Arabidopsis thaliana seedlings (biolistic transformation or transgenic Arabidopsis seedlings). 1. DNA containing the luciferase gene is transformed into the appropriate cells. 2. The tissue is put into 1.5 ml clear eppendorf tubes or 12 X 50 round bottom Polypropylene tubes containing 0.5 ml water. 3. To this 50 m l of coelenterazine substrate is added at a final concentration of 1m M. 4. Mixing is done by tapping and luminescence readings are taken within 30 seconds using luminometer. The readings obtained are in Relative Luminescence Units (RLU). The luminescence is measured after a 2 seconds delay and a 10 seconds integration period. 5. In order to do a time course one can also take readings after 1min, 2 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 after 30 minutes. 6. Replicate readings. If the instrument is connected to a computer then the readings are recorded on to a 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. 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): Blue: 333nm-463nm with >90% transmission from 370-410nm Yellow: 520nm longpass with >80% transmission from 550nm up These are Wratten gel filters. 2. Wallac plate reader: Blue: 460nm /50nm width (i.e 460+/-25nm; Chroma) Yellow: 535/40 (Chroma). (can also try 585/40 or 550 longpass). 3. Packard Fusion for standard BRET (built-in): Blue: 485/20 Yellow: 540/25 or 530/25 Packard Fusion for BRET2: Blue: 410 nm Yellow: 515 nm(use DeepBlueC as substrate).
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. 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. 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, 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. Buffer B is a high salt buffer suggested by Mayerhofer and Szalay, 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 upon addition of substrate. （from von Arnim lab，Please contact Dr. A. von Arnim with requests for vectors, procedures, or advice.）
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