Weak Chemiluminescent Western Blot Signals: Possible Cause 3 – Wrong Membrane Placement

How to Place the Blot on the C-DiGit Blot ScannerSo, we’ve 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 placement of the blot 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. The placement of the blot 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 Correctly Imaged Chemiluminescent Blot on C-DiGit Scanner Incorrectly Imaged Chemiluminescent Blot on C-DiGit Scanner
Conditions:
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, stay tuned to future blog posts. And if you would like to try some FREE Western Blot Analysis Software, download Image Studio Lite today!

Related posts:
Weak Signals on Chemiluminescent Western Blots: Possible Cause 1 – Substrate Rate of Reaction
Weak Signals on Chemiluminescent Westerns: Possible Cause 2 – Not Enough Substrate

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Weak Signals on Chemiluminescent Westerns: Possible Cause 2 – Not Enough Substrate

The amount of chemiluminescent Western blot substrate you use can make a big difference in the results that you get. If you do not add enough substrate to your blot, the light-generating luminol will be depleted, leading to fewer photons (light) being released. (For more information on light collection and the chemiluminescent reaction, read “Imaging Chemiluminescence by Scanning“.)

Below are the results of an experiment where we looked at the performance differences when incubating the blot in different volumes of SuperSignal® West Pico1. The three blots have the same LOD (2.5 μg/well); however, signal intensity varies. Blots were all imaged on the C-DiGit® Blot Scanner. lmages are normalized to the LUT (Lookup Table) of the optimal blot. (Read more about Image Studio Software or download FREE Image Studio Lite Western Blot Analysis Software.)

Optimal Blot Satisfactory Blot Unsatisfactory Blot
Images Optimal Blot for Substrate Amount Use Chemiluminescent Western showing Satisfactory Results for Substrate Amount Used Unsatisfactory Chemiluminescent Western blot results with low signal
Conditions:
Substrate SuperSignal West Pico1 SuperSignal West Pico1 SuperSignal West Pico1
Substrate Volume 3.0 mL substrate 1.5 mL substrate 0.75 mL substrate
Imaging Method
  • Substrate placed directly on C-DiGit Blot Scanner glass surface.
  • Membrane placed on substrate, 1-ply sheet protector on top, incubate 5 min.
  • Substrate placed directly on C-DiGit Blot Scanner glass surface.
  • Membrane placed on substrate, 1-ply sheet protector on top, incubate 5 min.
  • Substrate placed directly on C-DiGit Blot Scanner glass surface.
  • Membrane placed on substrate, 1-ply sheet protector on top, incubate 5 min.
  • Scan Setting High High High
    Performance Bright signal Moderate signal Low signal

    1 SuperSignal West Pico is comparable to WesternSure™ ULTRA Chemiluminescent Substrate.

    For more hints and tips, stay tuned to future blog posts.
    Related posts:
    Weak Signals on Chemiluminescent Western Blots: Possible Cause 1 – Substrate Rate of Reaction