Okay, I know, research budget money is tight and you want to make your reagents stretch as far as possible, but it really is not a good idea to dilute your chemiluminescent Western blotting substrate.
Why? It is 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 |  |  |
| Substrate | SuperSignal® West Dura1 | SuperSignal® West Dura1 |
| Dilution | 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:
- Possible Cause 1: Substrate Rate of Reaction
- Possible Cause 2: Not Enough Substrate
- Possible Cause 3: Wrong Membrane Placement
- Possible Cause 4: Blot Processing Time
- Possible Cause 5: Good Western Blot Image Signal Acquisition Relies on Uniformly Wet Western Blots
- Possible Cause 6: If Comparing Film and Digital Imagers, Expose Blot on Digital Imager First
- Possible Cause 7: Imager Sensitivity Settings May Affect Detection of Chemiluminescent Western Blot Signals
- Possible Cause 8: Chemiluminescent Western Blot Substrate Temperature Affects Signal Strength
- Possible Cause 9: Don’t Rush Substrate Incubation Time for Chemiluminescent Western Blots
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