Bio-Rad Bio-Dot® and Bio-Dot SF Microfiltration Apparatus - Manual (Page 25)

b. Hybridization was insufficient. Incorporate 10% dextran sulfate in the hybridization
mixture. This polymer effectively reduces the solvent volume, thereby increasing the
concentration of the solutes and enhancing hybridization.
c. Exposure time was insufficient. Increase the time of exposure.
d. Sample load was insufficient. Increase the sample load.
e. Probe concentration is too low. If low signal is accompanied by low background, then the
probe concentration can be increased.
f. Binding of nucleic acid to the membrane was incomplete. See Troubleshooting, Part II.
g. If no autoradiographic signal is seen, make sure the probe was denatured by heating to
100°C, exposure to 0.4 N NaOH, or by heating to 65°C for 5 minutes in 50% formamide
prior to hybridization.
2. Protein
a. Antigen binding was incomplete. See Troubleshooting, Part II.
b. Monoclonal antibodies may not recognize a denatured antigen. Assess the binding of
other monoclonal or polyclonal antibodies. Blot only native proteins.
c. The enzyme conjugate or the substrate is inactivated. Primary or secondary antibody is
inactive or nonsaturating. Test the enzyme, antibody, and substrate separately for activity.
Increase concentration of the first or second antibody. Eliminate the detergents from
reactions and washes. With HRP, avoid sodium azide, as it is a potent inhibitor of the
enzyme.
d. For labeled probes, exposure time was insufficient. Increase the time of exposure.
e. Antibody reaction times are insufficient. Increase reaction times.
f. Sample load was insufficient. Increase the concentration of antigen applied.
V. Nonspecific or Nonquantitative Detection
1. Protein
a. Monoclonal antibodies may react nonspecifically with SDS-denatured proteins. Compare
binding of other monoclonals or polyclonal antibodies. Blot native protein.
b. Concentration of the primary or secondary antibody is excessive. Increase the dilution of
the antibodies.
c. Primary or secondary antibody is contaminated with nonspecific or species cross-reactive
IgG. Use a purified IgG primary antibody fraction and affinity-purified blotting grade
secondary antibody.
d. Slow, gentle filtration is needed for complete optimal protein binding.
2. DNA/RNA
a. Probe is not pure.
b. Blocker shares common sequences with the probe. Assess different blockers. Use more
stringent washes.
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