Bio-Rad Mouse Typer Isotyping Kit - Manual (Page 9)

Fig. 1. Suggested format for sub-isotyping 10 samples
on 1 microplate.
7. Empty the plates of panel reagents and flick-wash 5x with
PBS-tween.
8. With the exception of column 1, fill the wells with diluted goat
anti-rabbit horseradish peroxidase conjugate, 100 µl/well. Cover
and incubate for 1 hour.
9. During the incubation with conjugate, prepare peroxidase
substrate solution (see Section 2.1).
10. Wash off unreacted conjugate solution by flick-washing the
plate 4x with PBS-tween. Wash an additional time with PBS.
Invert and tap the plate over paper toweling to rid the wells of
excess wash solution. Add 100 µl peroxidase substrate
solution. Positive reactions will appear immediately. Identifi-
cation of mouse sub-isotype can be assessed after a 10 to 30
minute room temperature incubation.
11. Stop color development by adding 100 µl/well 2% oxalic acid
color stopping solution. Results can be documented using the
Model 3550, 3550-UV, or Model 550 Microplate Reader at
415 nm.
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2
+
C
O
N
T
R
O
L
B
L
A
N
K
Samples 1-10
7
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