Weak Signals on Chemiluminescent Western Blots Could Be Caused by Substrate Rate of Reaction

Thursday, 7 May, 2015


  • troubleshooting chemiluminescent western blots
  • chemiluminescent substrate
  • troubleshooting western blot
  • westernsure substrates
  • substrate rate of reaction

    • Are 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 cause to consider 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 Imaging System and 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. 

    The table below details 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 BlotSatisfactory BlotUnsatisfactory Blot
    Images



    SubstrateSuperSignal West FemtoSuperSignal West Dura1SuperSignal West Pico
    Substrate Volume3.0 mL substrate3.0 mL substrate3.0 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 SettingHighHighHigh
    PerformanceLOD – 78 ngLOD – 312 ngLOD – 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!

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