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:

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