User Manual

d. Proteins 15,000 daltons may show decreased binding to 0.45 µm

nitrocellulose. Use the Zeta-Probe membrane or 0.2 µm nitrocellulose. Also,

glutaraldehyde fixation will increase retention of small proteins and peptides to

both nitrocellulose and the Zeta-Probe membrane.

f. Protein may be removed from nitrocellulose by SDS, NP-40, or Triton X-100. Use

Tween 20 in washes. Reduce concentrations or time of any SDS or NP-40 washes.

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 nicktranslation 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 nicktranslation.

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 or 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 Bio-Rad’s 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, Hemoglobin reacts with horseradish peroxidase; 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.