Good Western Blot Image Signal Acquisition Relies on Uniformly Wet Western Blots

Have you discovered the cause of the weak signals from your chemiluminescent Western blot yet? Well, let’s keep going. Here is another possible cause – the uniform wetness of the blot. It’s important to keep your Western blot membrane uniformly wet during the entire Western blot image acquisition.

Why does this matter? Well, if you don’t add enough substrate, the membrane will not stay wet, and there will be no enzymatic activity. And, that means no signal to detect.


  • Use more substrate prior to imaging
  • Do not completely blot off all of the substrate before imaging

For C-DiGit® Blot Scanner:

  • Wrap the blot in plastic wrap or cover with a plastic sheet protector
  • Incubate blot with substrate directly on scanner bed

Below is a table showing results of an experiment in which blots of varying degrees of wetness were imaged. You can clearly see that the wet blot and the damp blot give the best results. For both, the blots were protected from drying out by using a 1-ply sheet protector that was placed on top of the blot.

Optimal Blot Optimal Blot Unsatisfactory Blot
Images Optimal Chemiluminescent Wet Blot Optimal Chemiluminescent Damp Blot Unsatisfactory Image
Conditions: Wet blot Damp blot Dry blot
Imaging Method Imaged in 3.0 mL of SuperSignal® West Dura1 substrate placed on the scan bed of the C-DiGit Blot Scanner with 1-ply sheet protector on top. Excess SuperSignal® West Dura1 substrate removed, then imaged on the scan bed of the C-DiGit Blot Scanner with 1-ply sheet protector on top. Blot dried before imaging.
Performance LOD – 640 ng LOD – 640 ng LOD – None detected

1SuperSignal West Dura results are comparable to those obtained with WesternSure® PREMIUM Chemiluminescent Substrate.

We still have 5 more possible causes of weak signals in chemiluminescent Western blots to review, so stay tuned to future blog posts. And if you would like to try some FREE Western Blot Analysis Software, download Image Studio™ Lite today!

Watch this short video to see how to correctly place a Western blot on the C-DiGit Blot Scanner surface.

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