LacZ activity measurement.

Protocol for Lac-Z assay

>The 96-wells-plate is centrifuged at 1800 rpm, 2min, RT

>The culture supernatant can be collected to a new 96-wells for ELISA assay (about 150uL per well)

>Then, remove the rest medium, 50 μL/well lysis buffer (0.1% Triton X-100, 250 mM Tris, pH 8.0) is added to each well, the plate is frozen at -80°C and thawed at RT

>Add 50 μL/well PBS containing 0.5% BSA into each well

>Add 100 μL/well substrate solution (1mg/mL chlorophenolred β-d-galactopyranoside) dissolved in β-galactosidase buffer (60mM sodium dibasic phosphate buffer, pH8, 1mM magnesium sulfate, 10mM KCl, 50mM β-mercaptoethanol)

>The plate is incubated at 37°C for 12 to 18 hours until color development reached a proper level

>The intensity of color development was measured at 580 nm using a microtiter plate reader

Reagents

Chlorophenolred β-d-galactopyranoside: 100X stock in -20°C

β-galactosidase buffer: 1X in 4°C

Lysis buffer: 1X in 4°C

PBS containing 0.5% BSA: 1X in 4°C

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