Imaging - LI-COR C-DiGit User Manual

Chapter 3: Scanning Membranes
2) How do I apply the substrate?
Make sure the substrate is at room temperature before use. Substrate should be applied by fol-
lowing the manufacturer's suggested methods for the amount of substrate to use and the incub-
ation time prior to imaging. The substrate can be directly added to the sample side of the
membrane (either by pipetting the substrate on the sample surface, or by preincubating the
membrane in the substrate), allowed to incubate, and scanned without having to wrap the mem-
brane before scanning. Membranes can also be wrapped in a plastic covering prior to scanning.
3) The membrane dried during imaging. Can I apply more substrate and image again?
No. Applying more substrate to a dried blot will likely result in high background.
4) How do I keep the membrane from drying out?
In general, the membrane should not dry out in the amount of time it takes to complete a scan.
However, to maintain moisture on the membrane for an extended amount of time, place a
clear, flat plastic covering on top of the chemiluminescent Western blot to keep the substrate in
contact with the HRP enzyme and to prevent the blot from drying out. Membranes can also be
completely wrapped in plastic covering prior to scanning (see Scanning Membranes on
page 19). Make sure there is no plastic wrap extending out past the scanning surface and into
the outer lid seal.

Imaging

1) Can I wrap the blot in plastic wrap before imaging?
As long as the plastic is clear, clean, and wrinkle free (the plastic wrap may cause unwanted
background, especially if it is folded or handled roughly), the blot can be wrapped in plastic
before imaging. Note that plastic wrap will cause unwanted background; we recommend using
plastic wrap only if you must seal the membranes.
2) Why are the bands on my blot so light?
Use the Lookup Table (LUT) in Image Studio to adjust how data are mapped to the display
pixels of your computer screen.
You can view the Image LUTs as either slider bars (the default view, below left) or as histograms
overlaid with a curve (below right). To hide this view, click the double arrows in the top right
corner.
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