Don’t Rush Substrate Incubation Time for Chemiluminescent Western Blots

Substrate Incubation Time is Important!Five minutes can seem like a long time, especially when you are waiting to image your chemiluminescent Western blot. But it is really important that you follow the manufacturer’s recommendation for incubation time. Typically, this is five (5) minutes for optimal photon emission – for both film and digital imaging.

So, set the timer for 5 minutes, grab your iPhone® or iPod® – or the crossword, and relax until the buzzer goes off.

To test this, we imaged a chemiluminescent Western blot immediately after adding the chemiluminescent substrate and then imaged a blot where we waited 5 minutes – answered a few emails, looked at the news, and downloaded a new app – and THEN imaged the Western blot. As you can see, incubating allowed us to see more bands and gave much better Western blotting results.

Optimal Blot Unsatisfactory Blot
Images Optimal Blot - 5 Min Substrate Incubation Unsatisfactory Blot - No Incubation
Conditions:
Substrate SuperSignal® West Pico SuperSignal® West Pico
Incubated for 5 minutes No incubation
Substrate at room temperature Substrate at room temperature
Performance LOD – 2.5 µg LOD – 5 µg

So slow down, take a breath, and wait for your chemiluminescent Western blot substrate to incubate on your Western blot before imaging.

Here are some other blog posts on possible causes of weak chemiluminescent Western blot signals:

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