If you are trying to compare how the same chemiluminescent Western blot looks when imaged on a digital imager (like the C-DiGit® Blot Scanner) with how it will look when imaged on film, it is important to know that you should expose the blot to film BEFORE imaging on a digital imager.
Why does this matter? Digital imaging requires capturing the most photons being generated, which is typically immediately after a 5-minute chemiluminescent substrate incubation. Time may be more of an issue with some substrates.
For more information on how film and digial imaging compare, read Western Blot Analysis: Comparison of film and the C-DiGit Blot Scanner.
In Table 1 below, performance differences of a Western blot detected with SuperSignal® West Pico when the same blot is imaged over time. Blot was incubated 5 min in substrate before imaging on the C-DiGit Blot Scanner. Images are normalized to the LUT of the optimal blot.
| Table 1 | Optimal Blot | Unsatisfactory Blot | Unsatisfactory Blot |
|---|---|---|---|
| Images |  |  |  |
| Imaging Time | Immediately after incubation with SuperSignal® West Pico | 26 min after incubation | 51 min after incubation |
| Scan Setting | High | High | High |
| Performance | LOD – 625 ng, Signal – 338 | LOD – 625 ng, Signal – 114 | LOD – 625 ng, Signal – 32.2 |
In Table 2, Performance differences of a Western Blot detected with SuperSignal West Dura1 when the same blot is imaged over time. Blot was incubated 5 min in substrate before imaging on the C-DiGit Blot Scanner. Images are normalized to the LUT of the optimal blot.
| Table 2 | Optimal Blot | Satisfactory Blot | Satisfactory Blot |
|---|---|---|---|
| Images |  |  |  |
| Imaging Time | Immediately after incubation with SuperSignal West Dura | 24 min after incubation | 48 min after incubation |
| Scan Setting | High | High | High |
| Performance | LOD – 156 ng, Signal – 12,300 | LOD – 156 ng, Signal – 10,400 | LOD – 156 ng, Signal – 9,090 |
In Table 3, Performance differences of a Western Blot detected with SuperSignal West Femto when the same blot is imaged over time. Blot was incubated 5 min in substrate before imaging on the C-DiGit Blot Scanner. Images are linked to the LUT of the optimal blot.
| Table 3 | Optimal Blot | Satisfactory Blot | Satisfactory Blot |
|---|---|---|---|
| Images |  |  |  |
| Imaging Time | Immediately after incubation with SuperSignal West Femto | 24 min after incubation | 48 min after incubation |
| Scan Setting | High | High | High |
| Performance | LOD – 156 ng, Signal – 11,500 | LOD – 156 ng, Signal – 8,120 | LOD – 156 ng, Signal – 6,860 |
1Comparable to WesternSure® PREMIUM Chemiluminescent Substrate
More posts to help you troubleshoot 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 Blots Uniformly Wet
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