Primary And Secondary Antibodies - LI-COR C-DiGit User Manual

3. Can I dilute the HRP-conjugated secondary antibodies in Odyssey
blocking buffer?
No. Odyssey blocking buffer contains sodium azide as a preservative. Sodium
azide binds irreversibly to the HRP enzyme, inhibiting the binding of the
substrate and slowing the 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 containing 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 antigen 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 helpful. LI-COR offers the
WesternSure™ Goat anti-Mouse HRP secondary antibody under part number
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.
Using Membranes
C-DiGit User Manual
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