Lumi-Film Chemiluminescent Detection Film Troubleshooting

Product No. LUMFILM-RO

Troubleshooting

High background on only part of membrane

  1. Possible cause:
    Drying of membrane during chemiluminescent detection procedure. For an example, please refer to the attached document “High background Example 1”.

    Recommendation:
    Do not wrap membrane in plastic wrap during incubation with chemiluminescent substrate. Plastic wrap cannot be sealed and will allow membrane to dry out. Carefully seal the damp membrane (in a development folder or hybridization bag) during the incubation/exposure to X-ray film. Check the seals to make sure liquid cannot leak.

  2. Possible cause:
    Membrane dried while in Blocking solution and stuck to side of incubation tray. For an example please refer to the attached document “High background Example 2”.

    Recommendation:
    Never let membrane dry at any stage of the prehybridization, hybridization, or detection procedures. Always use enough liquid in each incubation to cover membrane completely. Control the membrane occasionally during incubations (especially those with agitation) to ensure it does not dry or stick to the incubation tray.

Unspecific signals and smear

Possible causes:
Inappropriate membrane handling during procedure.
Note: Every scratch, touch, or gouge on the surface of the membrane will be made visible during the chemiluminescent detection/visualization procedure.
For an example, please refer to the attached document “Unspecific signals and smear”. Blot that had previously been hybridized only with radioactively labeled probes was rehybridized with a DIG-labeled probe.
Note: All these background "marks" were not visible in the radioactive detection procedure.

Recommendation:
Be very careful when handling the blot during a DIG procedure. Handle it only by the edges, and only with gloves and forceps. Do not touch the experimental portion of the blot with anything. Always start with a fresh blot when performing a DIG procedure for the first time; do not reuse a blot from a previous radioactive detection procedure. Membrane damage that is invisible during a radioactive procedure may be visible in a DIG procedure.

Too weak or too strong signals

At first, this might be an indication that the chemiluminescent assay exposure time is either too short or too long. For an example please refer to the attached document “Too weak or too strong signals”. Therefore, as a first measure increase or decrease the time you expose the blot to X-ray film or in an imaging instrument.

Further reason for a too high signal may be:
Probe concentration was too high: Reduce the probe concentration. Never use the entire yield from a labeling reaction to a analyze a single blot. Rather, perform a mock hybridization with different concentrations of the probe first to determine the amount of probe that gives the most signal with the least background.

Further reasons for a too low signal may be:

  • Probe concentration too low: Increase probe concentration an/or hybridize overnight.
  • Target nucleic acid degraded: Check amount of target DNA/RNA and repeat blot with freshly prepared DNA/RNA.
  • Inefficient capillary transfer.
  • Wrong type of membrane: Use the function-tested nylon-membrane from Roche Applied Science.
  • Inefficient probe labeling: Check labeling efficiency. See the Troubleshooting section in the package insert for the specific DIG labeling kit you use.

Irregular, smeared, grainy background

Possible cause:
Non-uniform distribution of chemiluminescent substrate, e.g. due to performing incubation while membrane wrapped in plastic wrap. Wrinkles in the hybridization bag, causing uneven contact between membrane and X-ray film. Drying of membrane during chemiluminescent visualization procedure. For an example please refer to the attached document “Irregular smeared grainy background”.
Note: The grainy appearance of the background indicates that the drying occurred during the chemiluminescent procedure rather than during hybridization. Drying during hybridization leads to a cloudy background (as shown in Problem irregular and cloudy background).

Recommendation:
Do not wrap membrane in plastic wrap during incubation with chemiluminescent substrate. Spread the chemiluminescent substrate uniformly over the surface of the membrane. Before exposing bag to X-ray film, flatten any wrinkles between blot and membrane by rolling a pipette over the surface of the bag. Carefully seal the damp membrane (in a folder or bag) during the incubation/exposure to X-ray film. Check the seals to make sure liquid cannot leak.

Spots on X-ray Film

Possible cause:
Electrostatic charge on the outside of the sealed hybridization bag. For an example, please refer to the attached document “Spots on X-ray film”.

Recommendation:
Wipe the surface of the sealed bag with 70% ethanol before incubating it with the X-ray film. When handling the membrane, always wear gloves. Use forceps, never fingers, to grip the membrane. Grip only the edges of the membrane, never the center (even with the forceps).

Gray circles above bands

Possible cause:
Membrane was too dry before chemiluminescent substrate was added. The substrate dried at several spots on the membrane, leading to the gray circles. For an example please refer to the attached document “Gray circles above bands”

Recommendation:
Do not let the membrane dry (even slightly) before adding the chemiluminescent substrate. Cover the membrane with the second sheet of the folder or bag immediately after you add the chemiluminescent substrate. Even a little dried substrate can lead to gray circles (especially if you are using CDP-Star).

Materials

     
Related Links