( cell extracts (three vials) and buffer...)Use of BRET2 luminescence cell-based assays with LEADseeker Multimodality Imaging System

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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