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Advantages of NIR

Comparison of DDAOG and conventional ONPG beta-gal assays.

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

  • 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.
Linear relationship between signal intensity and cell number.

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).

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

  • Higher sensitivity than assays with ONPG substrate, due to improved signal-to-noise (see Figure 1)
  • 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 3)
Comparison of beta-gal/DDAOG with luciferase promoter assay.

Figure 3. 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).

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


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