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
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.
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
In this series of posts, you will discover how easy it is to use Image Studio™ Lite. This free Western blot analysis software from LI-COR® allows you to easily create your own work area and then import images from numerous sources. So, if you have an old film scan, or an image from another chemiluminescent Western blot imaging system, try using this free Western blot analysis software. In subsequent posts, we will talk about the other functionalities of Image Studio Lite.
This second video walks through the steps needed to import an image into Image Studio Lite. Image Studio Lite analyzes images in the tif, png, or jpeg format as well as images acquired with past versions of the Odyssey® or Pearl® imaging systems software.