The amount of chemiluminescent Western blot substrate you use can make a big difference in the results that you get. If you do not add enough substrate to your blot, the light-generating luminol will be depleted, leading to fewer photons (light) being released. (For more information on light collection and the chemiluminescent reaction, read Imaging Chemiluminescence by Scanning.)
Below are the results of an experiment where we looked at the performance differences when incubating the blot in different volumes of SuperSignal® West Pico. The three blots have the same LOD (2.5 μg/well); however, signal intensity varies.
Blots were all imaged on the C-DiGit® Blot Scanner. lmages are normalized to the LUT (Lookup Table) of the optimal blot. (Read more about Image Studio™ Software or download FREE Image Studio Lite Western Blot Quantification Software.)
| Optimal Blot | Satisfactory Blot | Unsatisfactory Blot | |
|---|---|---|---|
| Images |  |  |  |
| Substrate | SuperSignal West Pico | SuperSignal West Pico | SuperSignal West Pico |
| Substrate Volume | 3.0 mL substrate | 1.5 mL substrate | 0.75 mL substrate |
| Imaging Method |
|
|
|
| Scan Setting | High | High | High |
| Performance | Bright signal | Moderate signal | Low signal |
Look for other blog posts covering common problems and solutions for chemiluminescent Western blots. Or, refer to Good Westerns Gone Bad: Maximizing Sensitivity on Chemiluminescent Western Blots.
Related posts on troubleshooting chemiluminescent Western blots: Possible Cause 1: Substrate Rate of Reaction
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