The amount of chemiluminescent Western blot substrate you use can make a big difference in the results that you get. If you do not add enough substrate to your blot, the light-generating luminol will be depleted, leading to fewer photons (light) being released. (For more information on light collection and the chemiluminescent reaction, read “Imaging Chemiluminescence by Scanning“.)
Below are the results of an experiment where we looked at the performance differences when incubating the blot in different volumes of SuperSignal® West Pico. The three blots have the same LOD (2.5 μg/well); however, signal intensity varies. Blots were all imaged on the C-DiGit® Blot Scanner. lmages are normalized to the LUT (Lookup Table) of the optimal blot. (Read more about Image Studio™ Software or download FREE Image Studio Lite Western Blot Analysis Software.)
For more hints and tips, stay tuned to future blog posts.
Weak Signals on Chemiluminescent Western Blots: Possible Cause 1 – Substrate Rate of Reaction