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Use of BRET2 luminescence cell-based assays with LEADseeker Multimodality Imaging System

cell extracts (three vials) and buffer (four vials) should be thawed on ice.

1.2 Preparation of DeepBlueC

1.2.1 Add 25 µl ethanol to the DeepBlueC vial (final concentration = 1 mM).

1.2.2 Prepare a 100-fold dilution by adding 1.98 ml of buffer to 20 µl of the solution prepared in 1.2.1 (final concentration = 10 µM).

1.2.3 Place the solution on ice, protected from light.

2. Assay procedure
(Note: The assay was set up in triplicate and both 10 µl and 2 µl cell-extract assays were performed in the same microplate.)
2.1 Add 15 µl BRET2 buffer to wells A1–A3, B1–B3, and C1–C3.

2.2 Add 23 µl BRET2 buffer to wells A4–A6, B4–B6, and C4–C6.

2.3 Add 25 µl BRET2 buffer to wells D1–D3.

2.4 Dispense 10 µl of each cell extract as follows:

2.4.1 A1–A3: clear cap (non-transfected cells)

2.4.2 B1–B3: blue cap [9Rluc(h) + GFP2 transfected cells -negative BRET control]

2.4.3 C1–C3: green cap [GFP2 - Rluc(h) fusion transfected cells - positive BRET control]

2.5 Dispense 2 µl of each cell extract as follows:

2.5.1 A4–A6: clear cap (non-transfected cells)

2.5.2 B4–B6: blue cap [9Rluc(h) + GFP2 transfected cells -negative BRET control]

2.5.3 C4–C6: green cap [GFP2 - Rluc(h) fusion transfected cells - positive BRET control]

2.6 Dispense 25 µl 10 µM DeepBlueC (see Reagent Preparation Protocol, above) to all wells.

2.7 Image on LEADseeker Multimodality Imaging System for 60 s (within 60 min following dispensing).


Results
The BRET2 demonstration
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