Possible Cause 10 for Weak Chemiluminescent Western Blot Signals: Diluting Substrates

westernsure-premium-926-95000Okay, I know, research budget money is tight and you want to make your reagents stretch as far as possible, but it really not a good idea to dilute your chemiluminescent Western blotting substrate.

Why? It’s because the rate of reaction is determined by the ratio of enzyme to substrate. Diluting substrates will dramatically impact the overall generation of light. Then, you will have to repeat the experiment, and you end up using more substrate anyway!

Optimal Blot Unsatisfactory Blot
Images Optimal Western Blot - Substrate Not Diluted Unsatisfactory Chemiluminescent Western Blot - Substrate Diluted
Conditions:
Substrate SuperSignal® West Dura1 SuperSignal® West Dura1
Substrate NOT diluted. Substrate diluted 1:1 (in water)
Performance LOD – 1.25 µg LOD – 2.5 µg

1Comparable to WesternSure® PREMIUM Chemiluminescent Substrate

So don’t skimp – use the substrate full strength the first time to ensure that you are seeing all of your protein bands. Or you might just have to repeat the experiment (and that will just cost you more time and money. . .)!

Here are the other nine possible causes of weak chemiluminescent Western blot signals:

If Comparing Film and Digital Imagers, Expose Blot on Digital Imager First.

If you are trying to compare how the same chemiluminescent Western blot looks when imaged on a digital imager (like the C-DiGit® Blot Scanner) with how it will look when imaged on film, it’s important to know that you should expose the blot to film BEFORE imaging on a digital imager.

Why does this matter? Digital imaging requires capturing the most photons being generated, which is typically immediately after a 5-minute chemiluminescent substrate incubation. Time may be more of an issue with some substrates. For more information on how film and digial imaging compare, read Western Blot Analysis: Comparison of film and the C-DiGit Blot Scanner.

In Table 1 below, performance differences of a Western blot detected with SuperSignal® West Pico when the same blot is imaged over time. Blot was incubated 5 min in substrate before imaging on the C-DiGit Blot Scanner. Images are normalized to the LUT of the optimal blot.

Table 1 Optimal Blot Unsatisfactory Blot Unsatisfactory Blot
Images Optimal Blot with SuperSignal West Pico Unsatisfactory Blot with West Pico Unsatisfactory Blot with West Femto
Conditions: Immediately after incubation with SuperSignal West Pico 26 min after incubation 51 min after incubation
Imaging Time Immediately after incubation with SuperSignal® West Pico 26 min after incubation 51 min after incubation
Scan Setting High High High
Performance LOD – 625 ng, Signal – 338 LOD – 625 ng, Signal – 114 LOD – 625 ng, Signal – 32.2

In Table 2, Performance differences of a Western Blot detected with SuperSignal West Dura1 when the same blot is imaged over time. Blot was incubated 5 min in substrate before imaging on the C-DiGit Blot Scanner. Images are normalized to the LUT of the optimal blot.

Table 2 Optimal Blot Satisfactory Blot Satisfactory Blot
Images Optimal with West Dura Satisfactory with West Dura Satisfactory with West Dura
Conditions:
Imaging Time Immediately after incubation with SuperSignal West Dura 24 min after incubation 48 min after incubation
Scan Setting High High High
Performance LOD – 156 ng, Signal – 12,300 LOD – 156 ng, Signal – 10,400 LOD – 156 ng, Signal – 9,090

In Table 3, Performance differences of a Western Blot detected with SuperSignal West Femto when the same blot is imaged over time. Blot was incubated 5 min in substrate before imaging on the C-DiGit Blot Scanner. Images are linked to the LUT of the optimal blot.

Table 3 Optimal Blot Satisfactory Blot Satisfactory Blot
Images Optimal Blot with West Femto Satisfactory Blot with West Femto Satisfactory Blot with West Femto
Conditions:
Imaging Time Immediately after incubation with SuperSignal West Femto 24 min after incubation 48 min after incubation
Scan Setting High High High
Performance LOD – 156 ng, Signal – 11,500 LOD – 156 ng, Signal – 8,120 LOD – 156 ng, Signal – 6,860

1Comparable to WesternSure® PREMIUM Chemiluminescent Substrate

Related posts:

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!