Making sure that the sensitivity setting is optimal to capture the most signal from your chemiluminescent Western blot could be the difference between getting a good, strong signal or getting a signal that you can barely see. This is our possible cause 7 for weak chemiluminescent signals.
How can you avoid possible cause 7 for LI-COR chemiluminescent imagers? On the C-DiGit® Blot Scanner, use High Sensitivity setting (12-min scan) for more sensitive detection. On the Odyssey® Fc Dual-Mode Imaging System, use a longer integration time (up to 10 min). Why is this important? Well, digital imaging with the C-DiGit Blot Scanner or Odyssey Fc Imager will not generally reach a saturation point. Begin with a longer acquisition time to ensure best sensitivity, then optimize to shorter scan times.
In Table 1 below, we tested the performance differences of a Western blot detected with SuperSignal® West Dura on the C-DiGit Blot Scanner when the same blot is imaged on High Sensitivity (12 min scan) versus Standard Sensitivity (6 min scan). As you can see, the longer scan time and higher sensitivity make a big difference in the results.
|Table 1||Optimal Blot||Satisfactory Blot|
|Conditions:||SuperSignal West Dura1||SuperSignal West Dura|
|Sensitivity||High (12 min)||Standard (6 min)|
|Performance||Signal – 12,300||Signal – 5,030|
1Comparable to WesternSure® PREMIUM Chemiluminescent Substrate
So be sure to check your sensitivity settings before you scan!
- 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
- Good Western Blot Image Signal Acquisition Relies on Uniformly Wet Western Blots
- If Comparing Film and Digital Imagers, Expose Blot on Digital Imager First.