Microplate Assays for β-Galactosidase Activity

The E. coli lacZ gene, encoding β-D-galactosidase, is extensively used as a reporter gene for detecting the expression of recombinant fusion genes and for monitoring transfection efficiency in mammalian, yeast, and bacterial cells. Although mammalian cells do contain β-galactosidases, they are generally lysosomal enzymes with low pH optima and therefore exhibit low activity at neutral pH. Combining this with the fact that E. coli β-D-galactosidase has a high turnover rate, the enzyme can be detected at very low levels, making it a sensitive reporter of gene expression.

β-galactosidase assay kits

The fluorogenic substrate CUG is quite water-soluble and can be used over a wide range of concentrations for the sensitive detection of β-D-galactosidase activity. This substrate is several orders of magnitude more sensitive than commonly used chromogenic substrates.

β-galactosidase substrates

These alternative fluorogenic substrates provide a choice of excitation and emission wavelengths for the sensitive detection of β-D-galactosidase activity.

Fluorescein di-β-D-glucopyranoside

Fluorescein di-β-D-glucopyranoside is one of the most sensitive substrates for glucosidases. The nonfluorescent substrate is sequentially hydrolyzed by β-glucosidase, first to fluorescein monoglucoside and then to highly fluorescent fluorescein.

FluoReporter® LacZ/Galactrosidase Quantitation Kit
Example of a standard curve using the FluoReporter® LacZ/Galactrosidase Quantitation Kit.

Selection guides
  FluoReporter® lacZ/Galactosidase Quantitation Kit
Target β-galactosidase
Reporter CUG
Ex/Em (nm) 386/448
Live cell No
Lysate Yes
Enzyme prep Yes
Usage Limit of sensitivity 0.5 pg of β-gal
Components Includes substrate and reference standard
Format 1,000 tests in 96-well format
Protocol outline
  1. Place cell extract in wells.
  2. Add CUG working solution.
  3. Incubate.
  4. Add stop buffer.
  5. Measure fluorescence
Cat. No. F2905
  4-Methylumbelliferyl β-D-Galactopyranoside (MUG) Fluorescein Di-β-D-Galactopyranoside (FDG) Resorufin β-D-Galactopyranoside 9H-(1,3-Dichloro-9,9-Dimethylacridin-2-One-7-yl) β-D-Galactopyranoside (DDAO galactoside)
Target β-galactosidase β-galactosidase β-galactosidase β-galactosidase
Reporter MUG FDG Resorufin β-D-Galactopyranoside DDAO galactoside
Ex/Em (nm) 360/449 490/514 571/585 645/660
Live cell No Yes No No
Lysate Yes Yes Yes Yes
Enzyme prep Yes Yes Yes Yes
Usage Detection of β-galactoside activity in cell extracts The most sensitive green fluorogenic substrate for detecting β-galactoside Red fluorogenic substrate for detecting β-galactoside Far-red fluorogenic substrate for detecting β-galactoside
Components Enzyme substrate Enzyme substrate Enzyme substrate Enzyme substrate
Format 1 g 5 mg 25 mg 5 mg
Protocol outline
  1. Place cell extract in wells.
  2. Add substrate working solution.
  3. Incubate.
  4. Add stop buffer.
  5. Measure fluorescence.
  1. Place cell extract in wells.
  2. Add substrate working solution.
  3. Incubate.
  4. Add stop buffer.
  5. Measure fluorescence.
  1. Place cell extract in wells.
  2. Add substrate working solution.
  3. Incubate.
  4. Add stop buffer.
  5. Measure fluorescence.
  1. Place cell extract in wells.
  2. Add substrate working solution.
  3. Incubate.
  4. Add stop buffer.
  5. Measure fluorescence.
Cat. No. M1489MP F1179 R1159 D6488
  Fluorescein Di-β-D-Glucopyranoside (FDGlu)
Target Glucosidases
Reporter FDGlu
Ex/Em (nm) 490/514
Live cell Yes
Lysate Yes
Enzyme prep Yes
Usage Sensitive green fluorogenic substrate for detecting glucosidases
Components Enzyme substrate
Format 5 mg
Protocol outline
  1. Place cell extract in wells.
  2. Add substrate working solution.
  3. Incubate.
  4. Add stop buffer.
  5. Measure fluorescence.
Cat. No. F2881
For Research Use Only. Not for use in diagnostic procedures.