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!

    5 Technical Tips for Chemiluminescent Western Blotting Success with the C-DiGit® Scanner

    In this short video, Jessica talks about 5 tips to help ensure that imaging chemiluminescent Western blots on the C-DiGit Blot Scanner is a success – the first time and always!

    Here’s a recap of the tips of those five technical tips:

    1. Install Image Studio™ Software on your computer before connecting the C-DiGit Blot Scanner.
    2. Incubate using room temperature substrate.
    3. Wrap your blot so it stays wet during the scan.
      1. Remember to place your blot protein side down.
      2. If sensitivity is an issue, use WesternSure® PREMIUM or SuperSignal® West Femto Chemiluminescent Substrate.
    4. Start with high sensitivity scan for your first scan and then work from there.
    5. Image on the C-DiGit Scanner first and then exposure your film.

    C-DiGit Blot ScannerHappy Blotting!