Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits - Manual (Page 13)

21
Troubleshooting Guide (continued)
Probable Recommended
Problem Cause Solution
e. Primary antibody i. Loss of reac
is not specific or tivity may
does not recognize have occurred
denatured antigens during elec-
(common with trophoretic
monoclonals). transfer. Pre-
test the reactiv-
ity of the anti-
body against
the antigen by
a dot blot.
2. High background. a. Exposure time was i. Decrease
too long. exposure time.
b. PBST washes after i. The washes are
transfer were omit- critical to
ted or insufficient. reduce spotted
or blotchy
background
development.
c. Blocking was insuf- i. Increase the
ficient. time of the
blocking step
and/or the con-
centration of
blocker used.
20
Troubleshooting Guide (continued)
Probable Recommended
Problem Cause Solution
d. Wash stringency i. Tween-20 is
was insufficient. necessary in
wash steps to
reduce back-
ground. The
concentration
can be
increased up to
0.3% if back-
ground persists.
ii. Increase the
number and
length of
washes.
e. Second antibody i. Use the recom-
conjugate was used mended dilu
at an excessive con- tion,or deter-
centration. mine the opti-
mal dilution
experimentally.
f. Contamination i. Refer to the
occurred during instrument
transfer. instruction
manual for rec-
ommendations.
g. Insufficient number i. Increase
of DMSO/PBST number of
washes. wash steps
following
BAR.
4100130c.qxd 4/9/99 8:29 AM Page 20