User Manual

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.

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. Asses 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 primary or secondary 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

proteins.

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

speciescross-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.