Troubleshooting Chemiluminescent Western Blots: Possible Cause 4 for Weak Signals – Blot Processing

Sometimes life in the lab gets crazy, right? You are finishing a Western blot and you realize that you are supposed to be at an important lecture across campus in 10 min!! Or, your spouse calls to say that one of the kids needs to be picked up as soon as possible. Yikes! The challenge is that blots should be processed and detected on the same day. And, the secondary antibody should be incubated the day of imaging and fresh substrate added just before imaging. Is it that important to your results? Yes, it is and just to prove it, we did a few experiments.

In Table 1, we studied performance differences when the same blot is imaged immediately after processing vs. stored overnight dry and then imaged. In Table 2, we looked at performance differences when the same blot is imaged immediately after processing vs. stored overnight wet and then imaged. Blots in both tables were all imaged on the C-DiGit® Blot Scanner. (And, all images are normalized to the Lookup Tables (LUT) of the respective optimal blot.)

For both experiments, you can see that saving the blot to image the next day is not a very good choice. This is because the secondary antibody and/or the chemiluminescent Western blot substrate is not stable enough for acceptable photon emission when digitally images after the day it is applied.

Table 1 Optimal Blot Unsatisfactory Blot Unsatisfactory Blot
Images Optimal Chemiluminescent Western Blot Unsatisfactory Chemiluminescent Western Blot Unsatisfactory Chemiluminescent Western Blot
Conditions:
Substrate SuperSignal® West Dura1 SuperSignal West Dura1 SuperSignal West Dura1
Processing Time Same Day Next Day Next Day
Detection Process HRP secondary incubated, washed, and substrate added immediately before imaging. HRP secondary incubated, washed, and substrate added day before imaging. HRP secondary incubated, washed, and substrate added day before imaging, then re-incubated with HRP secondary and substrate added immediately before imaging.
Storage Conditions Blot stored overnight dry, at room temperature Blot stored overnight dry, at room temperature
Performance LOD – 640 ng LOD – None detected LOD – 1.25 μg
Table 2 Optimal Blot Unsatisfactory Blot Unsatisfactory Blot
Images Optimal Chemiluminescent Western Blot Unsatisfactory Optimal Chemiluminescent Western Blot Unsatisfactory Optimal Chemiluminescent Western Blot
Conditions:
Substrate SuperSignal® West Dura1 SuperSignal West Dura1 SuperSignal West Dura1
Process Time Same day Next day Next day
Detection Process HRP secondary incubated, washed, and substrate added immediately before imaging. HRP secondary incubated, washed, and substrate added day before imaging. HRP secondary incubated, washed, and substrate added day before imaging, then re-incubated with HRP secondary and substrate added immediately before imaging.
Storage Conditions Blot stored overnight wet in PBS, at room temperature Blot stored overnight wet in PBS, at room temperature
Performance LOD – 640 ng LOD – None detected LOD – 1.25 μg

1SuperSignal West Dura results are comparable to those obtained with WesternSure® PREMIUM Chemiluminescent Substrate.

For more hints and tips, stay tuned to future blog posts. And if you would like to try some FREE Western Blot Analysis Software, download Image Studio™ Lite today!

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