β-Gal Reporter Gene Assay, chemiluminescent

Protocol tips

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	Wash cells and add β-Gal Reporter Gene Assay, incubate for 30 min at RT. Add substrate reagent and incubate for 15-60 min at RT

Protocol tips
Wash cells and add β-Gal Reporter Gene Assay, incubate for 30 min at RT. Add substrate reagent and incubate for 15-60 min at RT

Publication protocol

About one million RAW 264.7 macrophages stably expressing control vector, wild-type PPE2 or ΔNLS-PPE2 were transfected with 2.5 μg inos-luc (Kind gift from Prof. E.D Chan, University of Colorado, Denver) and 0.5 μg pCDNA-LacZ plasmids using Lipofectamine LTX along with plus reagent (3 μg) and at 8 hour post-transfection, cells were stimulated with 3 μg/ml LPS plus 10 ng/ml IFN-γ. Cells harvested at 24 hour post-stimulation were lysed with reporter lysis buffer (Promega, Madison, WI, USA) and protein concentration was estimated with Micro BCATM Protein Assay Kit following the manufacturer’s instructions. About 50 μg protein in 20 μl lysis buffer was mixed with 100 μl of luciferase substrate reagent (Promega). The light emitted was measured using TD 20/20 luminometer (Turner Biosystems, CA, USA). For β-Galactosidase reporter assay, 100 μg of protein was used. The β-Galalactosidase was quantitated using β-Galactosidase reporter assay kit (Roche Applied Science) following the manufacturer’s protocol as described earlier45. The results were expressed as relative luciferase units normalized to β-Galalactosidase activity.

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for β-Gal Reporter Gene Assay, chemiluminescent below.

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