So, we have talked about how the substrate rate of reaction can cause weak Western blotting signals and how the amount of substrate used can affect signals on chemiluminescent Western blots. But, there are other possible causes of weak signals.
The third possible cause of weak signals is the blot membrane placement for imaging on the detection systems, since systems may vary as to how the blot should be placed on the scanning surface. Why is this important? Well, if the blot is placed incorrectly, you may or may not be able to visualize bands. If bands are visualized, they will be substantially reduced in signal.
As an example, LI-COR has two imaging systems for chemiluminescent Western blots: the Odyssey® Fc Dual-Mode Imaging System and the C-DiGit® Blot Scanner. Blot membrane placement depends on which one you use.
For the Odyssey Fc Dual-Mode Imaging System, the membrane needs to be placed FACE UP on the imaging tray. However, for the C-DiGit Blot Scanner, the membrane needs to be placed on the scanning surface FACE DOWN. (For a quick video demonstrating this, watch "How to Place Your Blot on the C-DiGit Blot Scanner".)
Below is an experiment we did to look at the performance differences between imaging the blot correctly (protein side down) and imaging the blot protein side up on the C-DiGit Scanner. (Images are normalized to the Lookup Table (LUT) of the correctly imaged blot.)
| Correctly Imaged Blot | Incorrectly Imaged Blot | |
|---|---|---|
| Images |  |  |
| Substrate | SuperSignal® West Dura1 | SuperSignal® West Dura1 |
| Imaging Method | Blot imaged protein side facing down | Blot imaged protein side facing up |
| Performance | LOD - 156 ng | LOD - 625 ng |
1SuperSignal West Dura results are comparable to those obtained with WesternSure® PREMIUM Chemiluminescent Substrate.
For more hints and tips, check out our other blog posts on chemiluminescent Western blotting tips. And, if you would like to try some FREE Western Blot quantification software, download Image Studio™ Lite today!
Related posts on troubleshooting chemiluminescent Western blots:
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