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 | |
|---|---|---|
| Images |  |  |
| Substrate | SuperSignal® West Pico | SuperSignal® West Pico |
| Substrate Temperature | Substrate at room temperature | Substrate cold |
| Sensitivity | Standard | Standard |
| 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!
More causes of weak signals on chemiluminescent Western blots:
- 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 Western Blot Uniformly Wet
- Possible Cause 6: Order of Imaging Is Important
- Possible Cause 7: Imager Sensitivity Settings Matter
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