Is Trying to Get into the Darkroom to Develop Your Western Blot Film Giving You Nightmares?

See your darkroom nightmares come to life in this short feature presentation from LI-COR. You never know what could be behind your darkroom door . . .

If you want to avoid future nightmares, check out the C-DiGit® Chemiluminescent Western Blot Scanner.

Everything you love about film, without the hassles – or the darkroom!!

Weak Signals on Chemiluminescent Westerns: Possible Cause 2 – Not Enough Substrate

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 Analysis Software.)

Optimal Blot Satisfactory Blot Unsatisfactory Blot
Images Optimal Blot for Substrate Amount Use Chemiluminescent Western showing Satisfactory Results for Substrate Amount Used Unsatisfactory Chemiluminescent Western blot results with low signal
Conditions:
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
  • Substrate placed directly on C-DiGit Blot Scanner glass surface.
  • Membrane placed on substrate, 1-ply sheet protector on top, incubate 5 min.
  • Substrate placed directly on C-DiGit Blot Scanner glass surface.
  • Membrane placed on substrate, 1-ply sheet protector on top, incubate 5 min.
  • Substrate placed directly on C-DiGit Blot Scanner glass surface.
  • Membrane placed on substrate, 1-ply sheet protector on top, incubate 5 min.
  • Scan Setting High High High
    Performance Bright signal Moderate signal Low signal

    For more hints and tips, stay tuned to future blog posts.
    Related posts:
    Weak Signals on Chemiluminescent Western Blots: Possible Cause 1 – Substrate Rate of Reaction