The temperature at which a chemiluminescent Western blot substrate is used can affect the strength of the signal that is captured from Western blot images. Really?? Absolutely! This is because enzyme activity is greatly reduced when it is cold. The substrate needs to be equilibrated to room temperature for digital imaging. This is true with film as well, but there may be a period of time after adding substrate and exposing to film during which the substrate has had a chance to equilibrate to room temperature.
In the table below, we show data from an experiment in which we tested the affect of temperature on Western blotting signal. For one blot, SuperSignal® West Pico chemiluminescent substrate was used right out of the refrigerator – cold, 4 °C. For the other blot, the chemiluminescent Western blot substrate was allowed to come to room temperature before digital imaging. As you can see the signal difference is quite large.
|Optimal Blot||Unsatisfactory Blot|
|Substrate||SuperSignal® West Pico||SuperSignal® West Pico|
|Substrate at room temperature||Substrate cold|
|Performance||Signal – 1,740||Signal – 200|
So make sure your substrate is at room temperature before using, especially when you are imaging with a digital imager!
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