- Coomassie Gels
- DNA Staining
- ELISA/FLISA
- EMSA/Gel Shift Assay
- Glycoprotein Detection
- In-Cell Western™ Assay
- In-Gel Western Assay
- Microwestern
- Northern Blot
- On-Cell Western Assay
- Protease Assay
- Protein Array
- Quantitative Western Blot
- Reporter Gene Assay
- Reverse Phase (Lysate) Array
- RNAi Screens
- Small Animal Imaging
- Southern Blot
- Tissue Section Imaging
- Transcription Factor Assay
APPLICATIONS for the
ODYSSEY® IMAGER
Applications for the Odyssey Imaging System
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).
[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).
[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).
[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).
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)
Techincal Resources
LI-COR Application Note:
NIR Fluorescent β-galactosidase Reporter Gene Assays Using LI-COR Odyssey
PubMed
-
Gong, H. et al.
β-Galactosidase activity assay using far-red-shifted fluorescent substrate DDAOG
Anal Biochem. 386(1): 59-64 (2009)
