Odyssey Imaging System
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APPLICATIONS FOR THE ODYSSEY

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REPORTER GENE ASSAY


Application Overview

β-gal assays can be performed directly with the Odyssey Imager in microplate format, using the near-infrared fluorescent substrate DDAOG (9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) β-D-galactopyranoside). The cleaved substrate fluoresces strongly in the 700 nm channel. This assay was published in Analytical Biochemistry in 20091.

The β-galactosidase (β-gal) reporter gene is widely used in genetics and molecular biology. Uses include:

  • Monitoring promoter activity in transfected cells

  • Analyzing protein interactions and translocation using enzyme fragment complementation

  • Monitoring tumor growth in animal models2-3 (poor cellular penetration by the substrate affects performance in animals)

Advantages of DDAOG fluorescent assay, compared to conventional β-gal assays:

  • Higher sensitivity than assays with ONPG substrate, due to improved signal-to-noise.

  • DDAO fluorescent signal is very stable.

  • Cell lysis and β-gal activity assay performed in a single buffer, making the assay simple and efficient.

  • pH and detergent concentration are important for optimal signal.

  • Similar performance to luciferase assays (see Figure 1).

1. Gong, H. et al. β-Galactosidase activity assay using far-red-shifted fluorescent substrate DDAOG Anal Biochem. 386(1): 59-64 (2009)

2. Buller, C.J. et al. Measurement of beta-galactosidase tissue levels in a tumor cell xenograft model, Methods Find Exp Clin Pharmacol. 25(9):713-6 (2003)

3. Tung, C.H. et al. In vivo imaging of beta-galactosidase activity using far red fluorescent switch. Cancer Res. 64(5):1579-83 (2004)

comparision bar graph

[LEFT] Figure 1. Comparison of β-gal/DDAOG with luciferase promoter assay. Activation of a transiently transfected reporter gene expression by MEKK is similar with the two systems.

Reprinted with permission from Gong, H. et al. Anal Biochem. 386(1): 59-64 (2009).

comparision line graph

[LEFT] Figure 2. Linear relationship between signal intensity and cell number. Signal produced by 10 µM DDAOG after incubation with cell lysate of different numbers of 9L/lacZ cells.

Reprinted with permission from Gong, H. et al. Anal Biochem. 386(1): 59-64 (2009).

comparision line graph

[LEFT] Figure 3. Comparison of DDAOG and conventional ONPG β-gal assays. Graphs show signal produced by cell lines with or without lacZ expression.  (A) DDAOG method; (B) conventional colorimetric ONPG method.

  • With DDAOG, fluorescent signal from 9L/lacZ cells was ~42-fold higher than the background from negative control cell lines.
  • With ONPG method, signal from 9L/lacZ cells was only ~3.5-fold higher than the background from negative controls.

Reprinted with permission from Gong, H. et al. Anal Biochem. 386(1): 59-64 (2009).