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[Detection of β-galactosidase by green fluorescence ]GlycoGREEN™-βGal

Features

  • Rapid & highly sensitive detection of β-galactosidase activity
  • Suitable for live cell imaging with minimal effects on cell growth.
  • Applicable to fixed cell staining and flow cytometry.

GlycoGREEN™-βGal is a fluorescent probe to detect enzymatic activity of β-galactosidase. Its green fluorescence can be used in combination with blue and red/orange fluorescent probes for multicolor imaging.

Applications

  • Detecting expression of lacZ reporter gene in living cells.
  • Detection of senescence (SA-βGal)
  • Detection of cancer with β-galactosidase expression

Principle of the measurement

GlycoGREEN™-βGal is a substrate for β-galactosidase. It has no fluorescence but fluoresces upon reaction with β-galactosidase. Generated fluorescent product is retained within cells.

Principle of the measurement

Features 1: Suitable for live cell imaging

High contrast images can be obtained during live cell imaging. GlycoGREEN™-βGal has quite low nonspecific fluorescence and after the reaction with β-galactosidase, it shows strong fluorescence. Besides, it has quite low toxicity even in >10× concentration for general use during imaging of cells.

Figure 1. Suitability for live cell imaging.
Figure 1. Suitability for live cell imaging.

Left – An example of live cell imaging. LacZ expressing HEK293 cells (LacZ+) and normal HEK293 cells (LacZ) are stained with 1 μM of GlycoGREEN™-βGal and another product S. Reagents were reacted at 37 ℃ for 15 minutes and observed under identical conditions.
Right –  Toxicity against ovarian cancer derived OVCAR5 cells. Cell viabilities in the presence of 0, 0.1, 1, 10, 100 µM of GlycoGREEN™-βGal were determined using MTT assay kit. GlycoGREEN™-βGal was reacted at 37℃ in 5% CO2 condition for 24 hours.

 
Features 2: Rapid & highly sensitive detection of β-galactosidase

GlycoGREEN™-βGal has almost no fluorescence but shows strong fluorescence of >100-fold after reaction with the enzyme β-galactosidase. The reaction is rapid under typical conditions of LacZ expressing cells, 15 minutes incubation of the cells with the reagent is sufficient to detect signals with high sensitivity.

※  More incubation time may be required depending on the expression level of β-galactosidase.

Figure 2. In vitro reaction time course of GlycoGREEN™-<i>β</i>Gal with <i>β</i>-galactosidase
Figure 2. In vitro reaction time course of GlycoGREEN™-βGal with β-galactosidase

β-galactosidase (final 5 units/mL) was added to 10 µM of GlycoGREEN™-βGal in phosphate buffer (pH 7.4, containing 0.1 % DMSO as a cosolvent). Fluorescence intensity change was measured using a microplate reader (TECAN infinite M200Pro) at 37℃. Excitation at 497 nm, emission, 519 nm.

Features 3: Applicable to fixed cell staining and flow cytometry.

Cell permeable GlycoGREEN™-βGal can detect β-galactosidase within cells. After reaction with β-galactosidase, generated fluorophore is retained within cells. Fluorescence was undiminished after fixation with 3% paraformaldehyde for 15 minutes.

Figure 3. Fluorescence microscopy of fixed cells using GlycoGREEN™-<i>β</i>Gal
Figure 3. Fluorescence microscopy of fixed cells using GlycoGREEN™-βGal

(left) Cells were fixed after the reaction with GlycoGREEN™-βGal (top) or other product S (bottom). After the reaction with 1 μM of reagents for 15 minutes, cells were fixed with 3% paraformaldehyde for 15 minutes. (right) Cells were fixed with 3% paraformaldehyde for 15 minutes and then, reacted with 1 μM of reagents for 15 minutes. Cells were rinsed before each observation. Cells were observed in a same condition.

GlycoGREEN™-βGal is also applicable to flow cytometric analysis.

Figure 4. An example of flow cytometric analyses
Figure 4. An example of flow cytometric analyses

Flow cytometry results of unstained HEK293 (LacZ+), HEK293 (LacZ) cells pre-reacted with 3 μM of AcidiFluor ORANGE (GC301) for 18 hours reacted with 1 μM of GlycoGREEN™-βGal for 1 hour, and HEK293 (LacZ+) cells reacted with 1 μM of GlycoGREEN™-βGal for 1 hour. Cells were washed with PBS and analyzed using FACSVerse (Becton Dickinson).

Spectra

Figure 5. Fluorescence and absorption spectra of GlycoGREEN™-<i>β</i>Gal
Figure 5. Fluorescence and absorption spectra of GlycoGREEN™-βGal

  • Spectra of 10 µM of GlycoGREEN™-βGal in phosphate buffer (pH 7.4, containing 0.1% DMSO as cosolvent). GlycoGREEN™-βGal has been reacted with β-galactosidase (5 units/mL) at 37℃ for 30 minutes.
  • For fluorescence measurement, the solution was excited at 497 nm.

References

Asanuma D, Sakabe M, Kamiya M, Yamamoto K, Hiratake J, Ogawa M, Kosaka N, Choyke PL, Nagano T, Kobayashi H, Urano Y  (2015)
Sensitive β-galactosidase-targeting fluorescence probe for visualizing small peritoneal metastatic tumours in vivo.
Nature Communications, 6:6463. doi: 10.1038/ncomms7463.

Price
Code No. Product Size Price Protocol MSDS
GC611 GlycoGREEN™-βGal 30 nmol×5 $398.00 Download
551KB
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257KB

Contact Information

Further information or questions on our products, please contact us, E-mail: info_itnl@goryochemical.com