If Comparing Film and Digital Imagers, Expose Blot on Digital Imager First.

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’s 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 Optimal Blot with SuperSignal West Pico Unsatisfactory Blot with West Pico Unsatisfactory Blot with West Femto
Conditions: Immediately after incubation with SuperSignal West Pico 26 min after incubation 51 min after incubation
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 Optimal with West Dura Satisfactory with West Dura Satisfactory with West Dura
Conditions:
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 Optimal Blot with West Femto Satisfactory Blot with West Femto Satisfactory Blot with West Femto
Conditions:
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

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