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 |  |  |
| Substrate | SuperSignal® West Pico | SuperSignal® West Pico |
| Substrate Temperature | Substrate at room temperature | Substrate at room temperature |
| Incubation Time | Incubated for 5 minutes | No incubation |
| 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.
For the complete information on troubleshooting chemiluminescent Western blots, refer to Good Westerns Gone Bad: Maximizing Sensitivity on Chemiluminescent Western Blots.
Related posts:
- Possible Cause 1: Substrate Rate of Reaction
- Possible Cause 2: Not Enough Substrate
- Possible Cause 3: Wrong Membrane Placement
- Possible Cause 4: Blot Processing Time
- Possible Cause 5: Keep Blots Uniformly Wet
- Possible Cause 6: Order of Imaging is Important
- Possible Cause 7: Imager Sensitivity Settings Matter
- Possible Cause 8: Substrate Temperature Affects Signal Strength
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