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 does not have a fast enough rate of reaction (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 Pico2
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
    2Comparable to WesternSure ULTRA 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!

    Exporting Data and Printing Lab Reports from LI-COR® Image Studio Lite Software

    WOO HOO! You are done analyzing your Western blot data with Image Studio Lite. SUPER JOB!!

    Now, I bet you want to export your data and create a lab report to give to your boss, right? Image Studio Lite offers the ability to export data for use in spreadsheet programs such as Microsoft Excel – and this video tutorial will walk you just how to do this.

    In this last video tutorial, you will learn how to print a lab report from Image Studio Lite software. You can create standardized or custom lab reports (add your institution logo, lab name, etc) that can be shared with colleagues — and your boss! — or used to create daily research logs.

    If at any time you need a refresher on how to use Image Studio Lite, you can view all of the tutorial videos and access all the support documents on our Image Studio Support page.

    Image Studio Lite – A Dream Come True, Without the Unicorn

    10 Possible Causes of Weak Signals on Chemiluminescent Western Blot Images

    Weak Signals on Chemiluminescent Western BlotsAre you seeing weaker than expected (hoped for. . .) signal on your chemiluminescent Western blot images with your digital imager? Not sure what could be causing this? Well, here is a list of 10 possible reasons why you might be seeing weak signals in chemiluminescent Western blot data:

    1. Thechemiluminescent substrate does not have a fast enough rate of reaction.
    2. Not enough substrate was added to the blot.
    3. Membrane was placed on the detection system incorrectly.
    4. Blot was not detected or processed on the same day it was imaged.
    5. Blot was not kept uniformly wet during the entire image acquisition.
    6. Blot was exposed to film BEFORE imaging on a digital imager.
    7. Blot was imaged using incorrect sensitivity setting (learn about the easy-to-use Image Studio Software. Try FREE Image Studio Lite Western Blot Analysis Software to see just how easy it is!)
    8. Chemiluminescent substrate was too cold.
    9. Chemiluminescent substrate was not incubated for 5 minutes.
    10. Substrate was diluted.

    Hum, that’s quite a list! For details on ways to eliminate or avoid these causes and get great results with your chemiluminescent Western blots, read Good Westerns Gone Bad: Maximizing Sensitivity on Chemiluminescent Western Blots.

    How Can I Set the Background and Adjust the Image Appearance in Image Studio Lite?

    Glad you asked! :-) So, you’ve started your Western blot analysis in Image Studio Lite. Great! (For a refresher, see Using Image Studio Lite, the Free Western Blot Analysis Software – Image Display and Analysis.) But what about setting the right background subtraction method? Hum. . . . .well, this short video tutorial will walk you through exactly how to do this.

    Background subtraction method selected – check!

    Okay, next you might want to adjust your Western blot image appearance. This can be done by using the Brightness/Contrast slider located on the right hand side of the screen. This quick video tutorial also shows you how you can do more advanced adjustments by using image Lookup histograms or Lookup tables.

    Wow! Your Western blot image analysis is looking pretty darn good! Great job!

    Have you told your friends about Image Studio Lite? Be sure to spread the word about this easy-to-use (and FREE!) Western blot analysis software. Just tell them to go right away to the Image Studio Lite web page and download their free copy. And, now that you have reviewed the tutorials so far – you can help them really get going fast!

    Next up – exporting data and printing lab reports.

    To see all of the tutorial videos and support documents, visit the Image Studio Support page.

    Image Studio Lite – A Dream Come True, Without the Unicorn