You do chemiluminescent Western blots – you do TONS of them. Sometimes, as I am sure you have discovered, you have ‘challenges’ – the darn thing just doesn’t look right!
What are some things that may happen when using chemiluminescence detection for Western blotting and how do you resolve them? The next series of blog posts here will offer up some tips on how to optimize chemiluminescent Westerns. These tips were designed for Westerns detected with a digital imager, like the Odyssey Fc Chemiluminescent and Near-infrared Fluorescent Imaging System. But many will also apply if you are (still) in the darkroom and using film. 🙂
Pools of excess substrate on the membrane can lead to areas of high background, as can adding more substrate to a membrane that has dried. Apply the substrate carefully and wick off any pools of substrate before imaging. Do not allow the membrane to dry.
The image above is an example of high background due to pooled excess substrate. If your blot looks like the image below, then the membrane dried out and then someone tried to add more substrate.