Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits - Manual (Page 11)

Section 4
Troubleshooting Guide
Probable Recommended
Problem Cause Solution
1. No reaction or a. Exposure time was i. Increase the
weak signal. too short. exposure time.
b. Primary antibody i. Store the anti-
solution is inactive body solution at
or non-saturating. the proper tem-
perature. Avoid
bacterial
contamination,
heat inactiva-
tion, and
repeated freeze-
thaw cycles.
ii. Antibody titer
was too low.
Increase the
concentration
of the antibody
used in the
assay.
iii.Tween-20 may
affect the reac-
tivity of some
antibodies.
Eliminate
Tween-20
from the assay
(except the
wash after the
blocking step).
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10. Wash 2x in PBST for 5 minutes each time.
11. Incubate membrane in diluted BAR for 10 minutes.
12. Wash 2–4x in 20% DMSO/PBST for 5 minutes each
time.
13. Wash 1–2x in PBST for 5 minutes each time.
14. Incubate membrane in diluted streptavidin-HRP for
30 minutes.
15. Wash 2x in PBST for 5 minutes each time.
Colorimetric Detection
16. Mix one part Opti-4CN diluent concentrate with nine
parts ddH
2
O. Prepare 0.25 ml per cm 2 of membrane.
17. Add 0.2 ml of Opti-4CN substrate per 10 mls of dilu-
ent. Mix well and pour onto membrane.
18. Incubate membrane with gentle agitation in the sub-
strate for up to 30 minutes or until the desired level of
sensitivity is attained.
19. Wash the membrane in ddH
2
O for 15 minutes.
20. Document or store membrane.
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