Luciferase complementation assays

The dual-color chck beetle luciferase complementation assay we describe is designed to quantify pair-wise interactions between two proteins, such as receptors CXCR4 and ACKR3, with a common binding partner. 

Luker, G., Luker, K. (2011). Luciferase protein complementation assays for bioluminescence imaging of cells and mice. Methods in Molecular Biology, 680, 29-43. 

Villalobos, V., Naik, S., Bruinsma, M., Dothaget, R., Pan, M., Samrakindi, M., etal. (2010). Dual-color click beetle luciferase heteroprotein fiagment complementation assays. Chemistry Biology, 17, 1018—1029. 

Upon lipopolysaccharide stimulation (1 tg/ml), Prabhu et al. (2002) found significantly higher iNOS transcript and protein expression levels with an increase in NO production in selenium-deficient RAW 264.7 cells than in the Se-complemented cells. Electrophoretic mobility-shift assays, nuclear factor-xB-luciferase reporter assays and Western blot analyses indicated that the increased expression of iNOS in Se deficiency could be due to an increased activation and consequent nuclear localization of the redox-sensitive transcription factor NF-xB. 

Plate cells at a density of 1 X 10 —2 X 10 cells per well in 100 pi complete growth medium with serum in black walled, clear bottom 96-well plates for tissue culture. Since complementation of click beetle luciferase fragments produces less bioluminescence than intact CBGN or red, we typically use 96-weIl rather than 384-weU plates for cell-based assays. This approach allows us to use shorter acquisition times in each emission channel and improves resolution for kinetics of signahng. For 384-weU plate assays, we use 3 x 10 —5 X 10 cells perweU. We typically culture cells for two days in 96-weU plates before assays. 

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