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|
|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:
- 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
- Possible Cause 5: Good Western Blot Image Signal Acquisition Relies on Uniformly Wet Western Blots
- Possible Cause 6: If Comparing Film and Digital Imagers, Expose Blot on Digital Imager First.
- Possible Cause 7: Imager Sensitivity Settings May Affect Detection of Chemiluminescent Western Blot Signals
- Possible Cause 8: Chemiluminescent Western Blot Substrate Temperature Affects Signal Strength on Western Blots
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