No Target Band

1. Primary/secondary antibody inactivation or improper species/antigen matching;

2. Incorrect electrophoresis polarity or membrane transfer orientation;

3. Excessive blocking or over-washing;

4. Expired luminescent substrate

1. Verify antibody specificity and expiration, store in aliquots to avoid repeated freeze-thaw;

2. Confirm correct assembly of electrophoresis and transfer components;

3. Optimize blocking (1-2h at RT with 5% non-fat milk/3% BSA) and washing (3×5-10min with TBST);

4. Use fresh, light-protected substrate

Weak Band Signal

1. Low protein concentration or incomplete extraction;

2. Insufficient transfer time/voltage;

3. Over-diluted antibodies;

 4. Inadequate incubation/exposure time

1. Increase sample amount or optimize extraction with inhibitors;

2. Adjust transfer parameters by protein molecular weight;

3. Follow antibody dilution recommendations (primary: 1:500-1:10000, secondary: 1:10000-1:50000);

4. Extend incubation (primary: 4℃ overnight) or exposure (1-5min)

High Background (Overall Membrane Glow)

1. Excess antibody concentration;

2. Inadequate blocking or incomplete washing;

3. Membrane drying during incubation/washing;

4. Excessive substrate or prolonged incubation

1. Reduce antibody concentration;

2. Extend blocking time (2h at RT) and increase washing frequency (4-5×10min with TBST);

3. Ensure membrane remains fully submerged;

4. Blot excess substrate and shorten incubation to 1-2min

Multiple Non-Specific Bands

1. Poor primary antibody specificity;

2. Protein degradation or cross-reactivity of secondary antibody;

3. Insufficient blocking/washing

1. Switch to monoclonal antibodies;

3. Add protease inhibitors during extraction and use species-matched secondary antibody;

3. Optimize blocking buffer (3% BSA for phosphorylated antigens) and washing conditions

Blurred/Tailed Bands

1. Protein aggregation/degradation;

2. High sample salt concentration or excessive loading;

3. Improper gel concentration or electrophoresis voltage

1. Avoid repeated freeze-thaw and add fresh reducing agents;

3. Desalt samples or reduce loading volume;

3. Select gel concentration based on protein size (8-12% for 30-100kDa) and use constant voltage (80V stacking gel, 120V resolving gel)

Abnormal Transfer

1. Insufficient/excessive transfer time/voltage;

2. Bubble formation in transfer sandwich;

3. Incorrect membrane pore size;

4. Abnormal methanol concentration in transfer buffer

1. Adjust transfer parameters (20-30min for <30kDa, 60-90min for >100kDa);

2. Remove bubbles during sandwich assembly;

3. Choose 0.2μm membrane for <20kDa, 0.45μm for others;

4. Maintain 20% methanol in transfer buffer

Uneven Bands (Spotted/Striped)

1. Uneven antibody distribution (insufficient shaking);

2. Precipitates in reagents;

3. Inadequate reagent volume covering the membra

1. Incubate on a shaker (50-100rpm);

3. Centrifuge reagents (10000rpm, 5min) to remove precipitates;

3. Ensure sufficient reagent volume (≥100μL/cm² membrane)