Reporter gene assay β-galactosidase substrates - Hep3B

Reporter gene assays enable high sensitivity measurement of gene expression and cell signaling through the addition of bioluminescent genes into target cells. One of the major challenges is to make a specific construct that has no responses other than those related to the signaling pathway of interest. This can be achieved by selecting highly specific reporter constructs containing only defined responsive elements and a minimal promoter linked to reporter enzymes such as luciferase

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Found 3 matching solutions for this experiment

Upstream tips
Seed 8 × 10^4 cells/well
Protocol tips
Dilute Galacton-Plus substrate 1:100 in Buffer B.

Add 25 μL of Buffer A to each well.

Within 10 min, inject 100 μL of Buffer B (containing Galacton-Plus substrate). After a 1-2 sec delay, read the luciferase signal for 0.1-1 sec/well

Incubate for 30-60 min at room temperature.
Upstream tips
Seed 2 × 10^4 cells and pretreat with agents.
Protocol tips
After72 h of pretreatment , fix cells with 2% paraformaldehyde/glutaraldehyde.

Add 1 ml of the Staining Mixture per well and incubate at 37 °C without CO2 until the cells are
stained blue
Protocol tips
Add 150µl of Assay 2X Buffer to sample.

Incubate the reactions at 37°C for 24 hours
Downstream tips
Read the absorbance at 420nm
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