Bio-Rad Mouse Typer Isotyping Kit - Manual (Page 10)

Note: When assessing color development using Model 3550,
Model 550, or the Model 3550-UV Microplate Reader, wash
the bottom of the assay plate thoroughly with distilled water
and wipe dry with non-lint toweling.
Section 3
Troubleshooting Guide
Problem Probable Cause Recommended Solution
A. High back- 1. Insufficient washing 1. Wash each well 6-7x and
ground. after conjugate anti- increase soak cycles to 30
body incubation. seconds.
2. Insufficient blocking 2. Increase blocking step to
after antigen adsorption. 60 minutes.
3. Tween 20 absent from 3. Include Tween 20 in all
washes. washes and solutions after
blocking.
4. GAR-HRP conjugate 4. Use recommended
concentration too high. dilutions. Generally, the
less dilute, the higher the
background.
5. Color developed too 5. Decrease color develop-
long. ment time by one-half.
6. Substrate too old (high 6. Use fresh solution A and
green color at working solution B
dilution).
7. Whole cell antigens 7a. Use extracted antigens.
have endogenous 7b. Use other enzyme con-
peroxidase activity. jugated antibodies.
7c. Destroy endogenous
activity by incubating
adsorbed Ag with mix-
ture of methanol/ H
2
O
2
(99 ml methanol, 1 ml
30% H
2
O
2
), 100 µl/well
for 1 hr.
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