Bio-Rad Mouse Typer Isotyping Kit - Manual (Page 11)

Problem Probable Cause Recommended Solution
B. No reaction or
weak color
development.
1. Horseradish a. Improper storage of a. Store peroxidase substrate
peroxidase sub- reagents. at 4 °C.
strate solution b. Substrate solutions hy- b. Use fresh peroxidase
inactive (Note 1). drolyzed due to age. substrate solutions.
2. Goat anti-rabbit a. Antibody improperly a. Store at 0-4 °C. Avoid
horseradish stored. bacterial contamination
peroxidase and repeated freeze thaw
conjugate is cycles.
inactive or non- b. Nonsaturating concen- b. If possible , increase
saturating
trations of monoclonal concentration of
(Note 2). or conjugate antibody monoclonal, conjugate, or
used in incubations. both. Use caution, the less
the conjugate is diluted
the greater the nonspecific
color produced.
3. Monoclonal anti- a. Monoclonal impro- a. Avoid bacterial contami-
body solution is perly stored. nation and heat inactiva-
inactive or non- tion.
saturating b. Antibody titer too low. b. Increase amount of hybri-
(note 3). doma culture media added
to plates (i.e. 200 µl).
May also be necessary to
increase conjugate anti-
body concentration.
c. Tween 20 deteriorates c. Eliminate Tween 20 from
reactivity of antibodies. all solutions and buffers
except wash after
blocking. Could result in
increased backgrounds.
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