Galacto-Star™ β-Galactosidase Reporter Gene Assay System for Mammalian Cells

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add diluted Galacton-Star® substrate 1:50 with Reaction Buffer Diluent. Add 100 µL of Reaction Buffer, mix, and incubate for 30-90 min until light emission is maxima	Measure light signal in a luminometer for 0.1-1 sec/well.

Protocol tips
Add diluted Galacton-Star® substrate 1:50 with Reaction Buffer Diluent. Add 100 µL of Reaction Buffer, mix, and incubate for 30-90 min until light emission is maxima
Downstream tips
Measure light signal in a luminometer for 0.1-1 sec/well.

Publication protocol

Cells were cotransfected with 0.1 μg wt or mt cyclin E-luciferase 26 or CDC2 promoter-reporter constructs and mammalian expression vectors for pCMV-β-Gal (10 ng), pCMV-Rb (300 ng), pCMV-E2F1/DP1 (10 ng), let-7 (100nM) and pCMV-AGO1 and -2 at the indicated amounts in ng using Lipofectamine 2000 (Invitrogen). β-galactosidase and luciferase activities were measured using Galacto-Star (Tropix) and Luciferase (Promega) luminescent assay kits according to the suppliers' instructions. Luciferase activities were normalized with β-galactosidase activities. Expression of proteins were confirmed by Western blot (data not shown). For siRNA, miR and antagomir transfection experiments, cells were transfected at 100nM. All siRNA duplexes were Smart Pools (Dharmacon). Pre-miR-let-7f, miR-let-7f and -let-7f-antagomir were purchased from Exiqon. Respective scrambled sequences were used as negative control.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Galacto-Star™ β-Galactosidase Reporter Gene Assay System for Mammalian Cells below.

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