6.2 High Background & Multiple Bands

Troubleshooting

1. Multiple Bands Appearing for the Target Protein
Rule out post-translational modifications, protein isoforms, and cleavage products by consulting protein databases and literature.
2. Sample-Related Causes
(1) Sample Degradation
Aliquot the sample immediately after preparation to minimize repeated freezing and thawing.
Add protease inhibitors to the sample.
Use freshly prepared samples.
(2) Excessive Sample Loading
Reduce the amount of protein loaded onto the gel.
(3) Inadequate Sample Denaturation
Add a sufficient amount of reducing agent (e.g., DTT or BME) during sample preparation.
Boil the sample for 5–10 minutes.
3. Antibody-Related Causes
(1) Non-Specific Binding of Primary Antibody
Decrease the concentration of the primary antibody.
Use 5% non-fat milk as the primary antibody dilution buffer (recommended).
Shorten the primary antibody incubation time.
Select high-quality monoclonal antibodies.
(2) Non-Specific Binding of Secondary Antibody
Identify non-specific binding of the secondary antibody by setting up a negative control (incubate with secondary antibody only, without primary antibody).
Decrease the concentration of the secondary antibody.
Use 5% non-fat milk as the secondary antibody dilution buffer (recommended).
Shorten the secondary antibody incubation time.
Select high-quality secondary antibodies.
4. Experimental Operation-Related Causes
(1) Insufficient Blocking
Replace the blocking buffer.
Increase the concentration of the blocking agent.
Extend the blocking time.
(2) Insufficient Washing
Add at least 0.1% Tween 20 to TBST (recommended).
Increase the duration and frequency of washing steps.
If the entire membrane shows a dark background, increase the NaCl concentration in TBST to 500 mM.
(3) Over-Exposure
Shorten the exposure time during chemiluminescence detection.
(4) Other Considerations
Prepare all buffers used in the experiment with double-distilled water (ddH₂O).
Ensure the membrane does not dry out during antibody incubation.
Use dedicated flat-tip forceps to handle and transfer the membrane; avoid creasing or scratching the membrane.
Do not reuse the filter papers used during the protein transfer step.