Primary And Secondary Antibodies; Washing Buffer; Substrates - LI-COR C-DiGit User Manual

chemiluminescent reaction. This results in less light production that may affect the appearance
of less intense bands or even the entire blot. For optimal results do not use any solutions con-
taining sodium azide for chemiluminescent Western blotting (for either secondary or primary
antibodies).
4) What is the best blocker for chemiluminescent Western blots?
It is best to try several blockers to find the one that gives the most satisfying data for each anti-
gen and antibody pair. There is not an optimum blocker for all conditions.

Primary and Secondary Antibodies

1) Why is the signal missing in the middle of the bands?
Too much secondary antibody on the membrane results in consumption of all of the substrate
in that area. Without substrate, there is no chemiluminescent signal and a blank spot appears in
the center of the band. Try different dilutions of the primary and secondary antibodies to find
what gives the best results, or try changing the substrate.
2) Does it matter where I purchase the HRP-conjugated secondary antibodies?
The reactivity of secondary antibodies ranges widely between vendors. The ratio of HRP enzyme
to antibody varies as well, and may affect the detection of the target. If the secondary antibodies
from one vendor are not working, trying antibodies from LI-COR or other vendors may be help-
ful. LI-COR offers the WesternSure
ber 926-80010, and the WesternSure Goat anti-Rabbit HRP secondary antibody under part
number 926-80011.
3) Should the HRP-conjugated secondary antibodies be highly cross- adsorbed?
Although highly cross-adsorbed antibodies are essential for two-channel, multiplex detection, it
is not always necessary with chemiluminescent blotting for a single target.

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 chemi-
luminescent 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, sub-
strates 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 sub-
strates that have been optimized to work with the C-DiGit Blot Scanner; two of these substrates
are WesternSure ECL (p/n 926-80100) and WesternSure PREMIUM (p/n 926-95000).
®
Goat anti-Mouse HRP secondary antibody under part num-
®
C-DiGit Blot Scanner
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