Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits User Manual

Page 13

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.

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.

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