β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 4.8×10^5 cells	Incubate for 90 min at 37 °C in 5% CO2.	Read absorbance at 420 nm

Upstream tips
Seed 4.8×10^5 cells
Protocol tips
Incubate for 90 min at 37 °C in 5% CO2.
Downstream tips
Read absorbance at 420 nm

Publication protocol

The day before transfection, 1.2×106 COS-7 cells are seeded in each 100-mm tissue culture dish, and 2×105 COS-7 cells are seeded in each well of 6-well plates.

For v-cells, 0.5 - 5 μg each of flipped VAMP plasmid is cotransfected with 5 μg of pTet-Off into the cells in each 100-mm culture dish. Control cells are cotransfected with the empty vector pcDNA3.1(+) and pTet-Off. To prevent the N-glycosylation of VAMPs 1, 4, 5, 7 and 8, tunicamycin (10 μg/ml) is included in cell culture medium during transfection.

For t-cells in each well of the 6-well plates, 1 μg of flipped SNAP-25 plasmid and pBI-G are cotransfected with 0.5 μg of flipped syntaxin1 plasmid or 0.05 μg of flipped syntaxin4 plasmid.

24 hr after transfection, the v-cells are detached from culture dishes with the Enzyme-free Cell Dissociation Buffer (Invitrogen). Detached cells are counted with a hemacytometer and resuspended in HEPES-buffered DMEM supplemented with 10% FBS, 6.7 μg/ml tunicamycin and 0.67 mM DTT.

4.8×105 resuspended v-cells are added to each well already containing the t-cells.

After 6, 12 or 24 hr rat 37 °C in 5% CO2, the expression of β-galactosidase is measured using the β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer according to the manufacturer's instructions (Promega). Briefly, the cells are washed twice with PBS, and then lysed in the Reporter Lysis Buffer. Cell lysates are mixed with equal volume of Assay 2× Buffer. As a blank control, the Reporter Lysis Buffer is mixed with the Assay 2× Buffer.

After 90 min, the colorimetric reaction is stopped by adding 1 M sodium carbonate.

Absorbance at 420 nm is measured using a HITACHI 100-40 spectrophotometer.

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Download the product protocol from Promega for β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer below.

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