Chemiluminescence is a dynamic, enzymatic process that introduces variability and error in your Western blot experiments. It’s often difficult to ﬁnd the “best” exposure, and the need for multiple exposures limits the reproducibility of your results.
Variability and error are introduced because:
- 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
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.
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 reproducibility of your Western blot data