Bio-Rad Bio-Dot® and Bio-Dot SF Microfiltration Apparatus - Manual (Page 24)

III. High Background After Incubation with Labeled Probes
1. DNA and RNA
a. Unincorporated label, small radioactive decay products, and small probe fragments
resulting from nick-translation can increase overall background. Use the Bio-Spin
®
chromatography columns to remove unincorporated label. Filter hybridization solutions
before use. Use the probe as soon as possible after preparation. Reduce exposure of the
probe to DNase during nick translation.
b. Improper blocking conditions were used. Increase the blocker concentration. Use a
different heterologous nucleic acid in the prehybridization mixtures. Sonicate the solution
thoroughly and denature before use.
c. The blocker shares common sequences with host or vector of cloned probe. Vary the
blocker. Yeast tRNA may be useful instead of salmon sperm DNA. Cut the probe out of
vector and purify.
d. Washes were insufficient. Include stringent washes; i.e., increase the temperature of the
washes, decrease the salt concentration. Increase the number and the length of the
standard washes.
e. The probe was too hot or concentrated. Dilute the probe.
f. The incubation period was too long. Shorten the reaction time.
g. The bag used in hybridization collapsed on the membrane. Be sure the membrane is
floating freely in the hybridization bag and that the volume of solution present is enough
to prevent the bag from collapsing during incubations.
h. Dust was present on the membrane. Remove by washing in 2x Denhardt’s prior to
baking or with a brief wash prior to hybridization.
i. The gasket is contaminated by radioactivity. Replace the gasket.
2. Protein
a. Impure secondary antibody was used. Use affinity purified blotting grade second
antibody.
b. Excessive reaction time in the substrate. Remove the blot from the substrate reaction
when the signal-to-noise level is acceptable.
c. Improper blocking conditions were used. Be sure the blocker is pure protein. Increase
the blocker concentration or blocking time. Match the blocker with the detection system;
i.e., hemoglobin reacts with horseradish peroxidase; bovine serum albumin (BSA) may
contain IgG contaminants.
d. Primary or secondary antibody is too concentrated. Dilute the antibodies.
e. Washes were insufficient. Increase the number and/or duration of the washes. Include
progressively stronger detergents in the washes. For example, SDS > NP–40 > Tween 20.
Also, include Tween 20 in the antibody buffers to reduce nonspecific binding.
IV. Poor Detection Sensitivity or No Reactivity
1. DNA/RNA
a. This problem may occur when total genomic DNA is probed for single copy or low copy
number genes. Try the Zeta-Probe membrane for binding and retention of increased
quantities of DNA.
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