LI-COR C-DiGit - Washing and Substrate Application

Using Membranes
3
C-DiGit User Manual 3-4
Washing Buffer
1. Does it matter how I wash the membranes after antibody incubation?
Yes. Adequately washing the membranes will greatly improve the appearance
of the chemiluminescent Western blot. Wash the membranes with a saline-
buffered solution containing 0.05 to 0.1% of a non-ionic detergent such as
Tween
®
20. Wash four times for five minutes each on a shaker or rotator with
ample wash solution.
Substrates
1. Which substrate should I use?
There are a wide variety of chemiluminescent substrates for HRP detection. In
general, substrates with a faster reaction rate tend to be more sensitive. The
C-DiGit Blot Scanner is designed to work well with most substrates. LI-COR
carries a line of chemiluminescent substrates that have been validated to work
with the C-DiGit Blot Scanner; two of these substrates are WesternSure™
ULTRA (p/n 926-85000) and WesternSure™ Premium (p/n 926-95000).
2. How do I apply the substrate?
Make sure the substrate is at room temperature before use. Substrate should be
applied by following the manufacturer’s suggested methods for the amount of
substrate to use and the incubation 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
membrane 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 back-
ground.
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 question 1 under
‘Imaging’ below). Make sure there is no plastic wrap extending out past the
scanning surface and into the outer lid seal.