Microplate Assays for β-Galactosidase Activity

The E. colilacZ 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.

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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
Example of a standard curve using the FluoReporter LacZ/Galactosidase Quantitation Kit.
Selection guides
 FluoReporter lacZ/Galactosidase Quantitation Kit
Targetβ-galactosidase
ReporterCUG
Ex/Em (nm)386/448
Live cellNo
LysateYes
Enzyme prepYes
UsageLimit of sensitivity 0.5 pg of β-gal
ComponentsIncludes substrate and reference standard
Format1,000 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
 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)
Targetβ-galactosidaseβ-galactosidaseβ-galactosidase
ReporterFDGResorufin β-D-GalactopyranosideDDAO galactoside
Ex/Em (nm)490/514571/585645/660
Live cellYesNoNo
LysateYesYesYes
Enzyme prepYesYesYes
UsageThe most sensitive green fluorogenic substrate for detecting β-galactosideRed fluorogenic substrate for detecting β-galactosideFar-red fluorogenic substrate for detecting β-galactoside
ComponentsEnzyme substrateEnzyme substrateEnzyme substrate
Format5 mg25 mg5 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.
Cat. No.F1179R1159D6488
 Fluorescein Di-β-D-Glucopyranoside (FDGlu)
TargetGlucosidases
ReporterFDGlu
Ex/Em (nm)490/514
Live cellYes
LysateYes
Enzyme prepYes
UsageSensitive green fluorogenic substrate for detecting glucosidases
ComponentsEnzyme substrate
Format5 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
β-galactosidase assay kits
 FluoReporter lacZ/Galactosidase Quantitation Kit
Targetβ-galactosidase
ReporterCUG
Ex/Em (nm)386/448
Live cellNo
LysateYes
Enzyme prepYes
UsageLimit of sensitivity 0.5 pg of β-gal
ComponentsIncludes substrate and reference standard
Format1,000 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
β-galactosidase substrates
 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)
Targetβ-galactosidaseβ-galactosidaseβ-galactosidase
ReporterFDGResorufin β-D-GalactopyranosideDDAO galactoside
Ex/Em (nm)490/514571/585645/660
Live cellYesNoNo
LysateYesYesYes
Enzyme prepYesYesYes
UsageThe most sensitive green fluorogenic substrate for detecting β-galactosideRed fluorogenic substrate for detecting β-galactosideFar-red fluorogenic substrate for detecting β-galactoside
ComponentsEnzyme substrateEnzyme substrateEnzyme substrate
Format5 mg25 mg5 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.
Cat. No.F1179R1159D6488
Glucosidase substrates
 Fluorescein Di-β-D-Glucopyranoside (FDGlu)
TargetGlucosidases
ReporterFDGlu
Ex/Em (nm)490/514
Live cellYes
LysateYes
Enzyme prepYes
UsageSensitive green fluorogenic substrate for detecting glucosidases
ComponentsEnzyme substrate
Format5 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

Expertly detect fluorescence with Thermo Scientific plate readers

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High-sensitivity fluorescence detection for 6-1,536 samples can be quickly performed on the Varioskan ALF or Varioskan LUX Multimode Microplate Reader using Invitrogen reagents to enable optimal detection. Take advantage of automatic dynamic range selection to get optimal gain settings for each individual well and automation capabilities for even higher throughput.

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