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 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 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

Weak Signals on Chemiluminescent Western Blots: Possible Cause 1 – Substrate Rate of Reaction

Optimal Chemiluminescent Western BlotAre you seeing weak signal in your chemiluminescent Western blot data? As we pointed out in a previous blog post, there are 10 possible reasons why this may be happening. Here is the first in our series on the causes and possible solutions/prevention measures you can try to get the best Western blot imaging data you can from your digital imager! We used our Odyssey® Fc Dual-Mode Imaging System and the newest member of our imaging family, the C-DiGit® Blot Scanner, in these studies.

Possible cause 1: Substrate rate of reaction is not fast enough (e.g., SuperSignal® West Pico)

Solution: Use WesternSure® PREMIUM or SuperSignal West Femto substrates

Why this matters: Different substrates have different rates of reaction. Some are developed to give off a lot of light quickly; others give off small amounts of light over longer periods of time. An alternate substrate may be required for digital imaging when imaging blots with low protein abundance.

Performance differences of three different substrate classifications using C-DiGit® Blot Scanner. All images are normalized to the Lookup Table (LUT) settings of the optimal blot for accurate visual comparison. (Learn more about easy-to-use Image Studio™ Software.)

Optimal Blot Satisfactory Blot Unsatisfactory Blot
Images Optimal Chemiluminescent Western Blot Satisfactory Chemiluminescent Western Blot Unsatisfactory Chemiluminescent Western Blot
Conditions:
Substrate SuperSignal West Femto SuperSignal West Dura1 SuperSignal West Pico
Substrate Volume 3.0 mL substrate 3.0 mL substrate 3.0 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 LOD – 78 ng LOD – 312 ng LOD – 2.5 μg

    1Comparable to WesternSure PREMIUM Chemiluminescent Substrate

    If you want to read ahead and find out ways to eliminate or avoid the other 9 causes of weak signals on chemiluminescent Western blots, read Good Westerns Gone Bad: Maximizing Sensitivity on Chemiluminescent Western Blots. Otherwise, stay tuned for more posts right here!

    No Darkroom? No Problem! Get the LI-COR® C-DiGit® Western Blot Scanner !

    C-DiGit Blot Scanner - a Compact Personal Chemiluminescent Imaging System
    Are you doing chemiluminescent Western blots? Have you ever found yourself with a blot that is ready to image, but the darkroom is busy or the developer is broken?

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    Watch this short video and then visit our website to get your very own C-DiGit Blot Scanner!