Is Your Chemiluminescent Western Blot Imaging Method a Source of Error and Variability?

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

Film Imager B Odyssey® Fc Imager
film usable range imager b usable range odyssey fc usable range
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 reproducibility of your Western blot data

Film and CCD Imaging of Western Blots: Exposure Time, Signal Saturation, and Linear Dynamic Range

Create a Work Area and Import an Image into this Free Western Blot Analysis Software

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.

The first video in this post will show you how to open Image Studio Lite software and create a work area. (If you need to find out the installation requirements for Image Studio Lite, see the 17-Jul-13 Post, Computer Requirements to Get Started with Free LI-COR® Image Studio Lite Software).

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.

Download FREE Image Studio Lite Western Blot Analysis Software from LI-COR and get started right away! (Bonus points for telling your friends about it too! :-))

Image Studio Lite – A Dream Come True without the Unicorn!