Digital Western Blots – Chemiluminescent Substrate Selection is Critical

Chemiluminescent Western BlotSo, you’ve selected your primary antibodies with care (see Know Thy Primary Antibody), and you’re using a great HRP-conjugated secondary antibodies. NOW – what about the chemiluminescent substrate?

Yes, I know, there are tons of different substrates and vendors out there – all claiming to be the best, right? So, how do you choose the right one for your chemiluminescence Western blot?

One thing to keep in mind is that in this wide variety of chemiluminescent substrates for HRP detection, there are some that are better suited for digital Western imaging than others. In general, choose a substrate with a faster rate of reaction for use with the Odyssey Fc Dual-Mode Chemiluminescent and IR Fluorescent Imaging System or other digital chemiluminescent imaging systems.

Some substrates that are designed for optimal performance on film may not be suitable for detection on a CCD-based imaging system. Try different substrates to find the one that gives the most desirable image. As you can see from the images below, the substrate you pick DOES make a difference! So choose carefully!

In the three images below, two-fold serial dilutions of HRP-conjugated secondary antibody (1 ng-1.25 fg) were spotted onto nitrocellulose using a slot blot apparatus. Blots were detected with various chemiluminescent substrates.

Chemiluminescent Substrate Comparison

Here are 2 documents with more troubleshooting information:

Updated February 18, 2015.