Luminescent β-galactosidase Detection Kit II

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Warm enough Reaction Buffer and Reaction Substrate for the entire experiment to room temperature	Add 200 µl of the Reaction Buffer Mixture to each cell lysate and mix gently. Incubate at room temperature (20–25°C) for 60 min

Upstream tips
Warm enough Reaction Buffer and Reaction Substrate for the entire experiment to room temperature
Protocol tips
Add 200 µl of the Reaction Buffer Mixture to each cell lysate and mix gently. Incubate at room temperature (20–25°C) for 60 min

Publication protocol

"TK-β-gal was a gift from B.L. Black (University of California San Francisco, San Francisco, CA). ACNS1 sequence (mm9, chr10:77509283-77509327) or ACNS1 with mutated kB sites (same mutations as in EMSA probes) was cloned into Kpn-linearized plasmid using standard cloning methods. p52-FLAG pcDNA3 was a gift from S. Smale (plasmid #20019; Addgene). RelB-FLAG pcDNA was also a gift from S. Smale (plasmid #20017; Addgene). pRL-CMV was purchased from Promega. 293T cells were cultured in high-glucose DMEM, 10% heatinactivated FCS, an penicillin/streptomycin, at 37°C, 10% CO2. 293T cells in 24-well-plates were transfected with 12.5 ng pRL-CMV and 250 ng of β-gal, p52, and RelB plasmids using Trans-IT 293 (Mirus). After 48 h, cells were briefly washed and lysed per Renilla Luciferase Assay System (Promega). Samples were then aliquoted an analyzed for Renilla luciferase using Renilla Luciferase Assay System and for β-gal activity using Luminescent β-galactosidase Detection kit II (Takara Bio Inc.) and luminescence measured using Victor2 1420 Multilabel Counter (PerkinElmer). In each experiment, all conditions were performed in triplicate."


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Manufacturer protocol

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