Have you discovered the cause of the weak signals from your chemiluminescent Western blot yet? Well, let’s keep going. Here is another possible cause – the uniform wetness of the blot. It’s important to keep your Western blot membrane uniformly wet during the entire Western blot image acquisition.
Why does this matter? Well, if you don’t add enough substrate, the membrane will not stay wet, and there will be no enzymatic activity. And, that means no signal to detect.
- Use more substrate prior to imaging
- Do not completely blot off all of the substrate before imaging
- Wrap the blot in plastic wrap or cover with a plastic sheet protector
- Incubate blot with substrate directly on scanner bed
Below is a table showing results of an experiment in which blots of varying degrees of wetness were imaged. You can clearly see that the wet blot and the damp blot give the best results. For both, the blots were protected from drying out by using a 1-ply sheet protector that was placed on top of the blot.
1SuperSignal West Dura results are comparable to those obtained with WesternSure® PREMIUM Chemiluminescent Substrate.
We still have 5 more possible causes of weak signals in chemiluminescent Western blots to review, so stay tuned to future blog posts. And if you would like to try some FREE Western Blot Analysis Software, download Image Studio™ Lite today!
Watch this short video to see how to correctly place a Western blot on the C-DiGit Blot Scanner surface.
- Weak Signals on Chemiluminescent Western Blots: Possible Cause 1 – Substrate Rate of Reaction
- Weak Signals on Chemiluminescent Westerns: Possible Cause 2 – Not Enough Substrate
- Weak Chemiluminescent Western Blot Signals: Possible Cause 3 – Wrong Membrane Placement
- Troubleshooting Chemiluminescent Western Blots: Possible Cause 4 for Weak Signals – Blot Processing