Chemiluminescence is a dynamic, enzymatic process that introduces variability and error in your Western blot experiments. It is often difficult to find the “best” exposure, and the need for multiple exposures limits the reproducibility of your results.
Sources of Variability and Error
- Chemiluminescent reaction changes constantly. The “best” exposure time is a moving target, so you must optimize and double-check every experiment.
- Multiple exposures are required. Common detection methods cannot accurately capture both faint and strong signals at once, without signal saturation.
Usable Data for Each Detection Method
| Film | Imager B | Odyssey® Fc Imager |
|---|---|---|
 |  |  |
| RESULT: Exposure time dramatically affects data output. Multiple exposures are required to detect strong and faint signals. Signal saturation cannot be determined visually. | RESULT: Multiple exposures are required to capture the full range of data. Strong signals are saturated (shown in blue). | RESULT: Multiple exposures are not required, because all exposure times yield consistent results. All data are captured in a single exposure without saturation. |
In the figure above, film was compared with a conventional, commercially-available CCD imager (Imager B), and the Odyssey Fc imager.
To eliminate variability introduced by blotting and chemiluminescent detection chemistry, a Harta luminometer reference plate (standardized light source) was used in place of a Western blot.
Conclusions
The Odyssey Fc imager outperformed both film and Imager B. All signals, from faintest to strongest, were detected – regardless of exposure time in a single exposure. No signal saturation occurred and all signals could be quantified.
With film and Imager B, however, longer exposures are needed to detect faint signals. In addition, stronger signals become saturated and cannot be quantified.
Choosing the Odyssey Fc Imaging System as your imaging method reduces variability and error in chemiluminescent Western blotting by giving you:
- All your data in a single exposure
- More reproducible results
- Simplified data analysis
Read the full study to learn:
- How chemiluminescence detection introduces variability and error
- How you can improve the repeatability of your Western blot data
Read: Film and CCD Imaging of Western Blots: Exposure Time, Signal Saturation, and Linear Dynamic Range
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