Failure Case Library

Real lab failures, root causes, and fixes — curated and bilingually annotated by our team.

All Techniques (7) Nucleic Acid Quantification (9) Lipid Transfection (8) End-point PCR Primers (9) Cell Culture (Contamination) (7) PCR (Sigma Guide) (12) Flow Cytometry (Controls) (8) PCR / qPCR Plastics (9) PCR (Invitrogen Guide) (8) Cell Culture (Precipitates) (6) Cell Culture (Cell Death) (8) ELISA (Sigma Guide) (6) PCR / RT-PCR Amplification Problems (5) Flow Cytometry (Paraformaldehyde Fixation) (14) Flow Cytometry (Sample Considerations) (14) ELISA (R&D Guide) (10) Western Blot Immunodetection (19) Flow Cytometry (CST Guide) (8) Immunohistochemistry (CST Guide) (14) Immunoprecipitation (CST Guide) (14) ChIP (CST Guide) (8) ELISA Development (9) Western Blot (Sigma Protocol) (8) IP-Western Blot (6) Western Blot (CST Guide) (8) Flow Cytometry (Fixation Buffers) (7) Flow Cytometry (Isotype Controls) (7) Flow Cytometry (Fixation & Permeabilization) (9) Flow Cytometry (Fc Blocking) (7) Flow Cytometry (Compensation) (7) Flow Cytometry (Autofluorescence) (7) Flow Cytometry (Troubleshooting) (8) Tissue Imaging (Autofluorescence) (9) ELISPOT (8) Immunoprecipitation (Protein Obstruction) (1) Immunoprecipitation (No Protein Detected) (5) Immunoprecipitation (High Background) (8) Immunoprecipitation (High Antibody Elution) (1) Immunohistochemistry (No Staining) (9) Immunohistochemistry (High Background) (9) ChIP (PCR Amplification Problems) (4) ChIP (Low Signal) (8) ChIP (Low Resolution with High Background) (6) ChIP (High Background) (1) Western Blot (Blue Background) (1) Western Blot (Unusual Gel Band Appearance) (3) Western Blot (Unexpected Multiple Bands) (7) Western Blot (Misshapen / Uneven Bands) (5) Western Blot (Bands at Wrong MW) (5) Western Blot (Detection Problems) (7) Western Blot (Weak / No Signal) (6) ELISA (Standard Curve Fit Problems) (6) ELISA (Inconsistent Results / High CV) (6) ELISA (High Background) (8) ELISA (Signal Problems) (11) ELISA (Competitive) (18) Cell-free DNA Extraction (8) Bacterial rRNA Depletion (4) RNA Depletion for RNA-seq (7) LAMP (Loop-mediated Isothermal Amplification) (7) HMW DNA Extraction (Monarch) (7) NGS Library Preparation (NEBNext Ultra II) (7) RNA Cleanup (4) DNA Cleanup & Plasmid Purification (7) PCR (Polymerase Chain Reaction) (26) Restriction Enzyme Digest (13) Western Blot (9) Immunohistochemistry (IHC) (6) Colony Formation Assay (10) Transwell Migration / Invasion Assay (5) qPCR (RT-qPCR) (5) Transfection (8) Plasmid Construction (1) Sanger Sequencing (2) Plasmid Mini-prep (1) Plasmid Construction (Double Digest) (1) Protein Extraction (4) Transfection (Co-transfection) (1) Transfection (siRNA Knockdown) (1) CCK-8 Cell Viability Assay (5) Wound Healing Assay (5)
Western Blot (Unexpected Multiple Bands) severe

Multiple Low Molecular Weight Bands or Smearing Below Expected Band

Multiple bands appear at molecular weights lower than the expected target protein, often accompanied by smearing or streaking below the primary band. This pattern suggests progressive breakdown of the intact protein.

💡 4 causes ✓ 4 fixes
Western Blot (Unexpected Multiple Bands) moderate

Multiple Bands or Smear Above Expected Molecular Weight

Multiple bands or a smear/streak appear at molecular weights higher than the predicted size of the target protein. This pattern is characteristic of post-translational modifications adding mass to the protein.

💡 4 causes ✓ 4 fixes
Western Blot (Unexpected Multiple Bands) minor

Bands at Lower Molecular Weight from Protein Cleavage

Distinct bands appear at molecular weights corresponding to known cleavage products or activated forms of the protein, rather than the full-length form. The fragment pattern may be specific and reproducible, distinct from degradation smearing.

💡 4 causes ✓ 4 fixes
Western Blot (Unexpected Multiple Bands) severe

Non-Specific Bands from Antibody Cross-Reactivity

Multiple bands appear at unexpected molecular weights that do not correspond to known forms of the target protein. These bands may vary in intensity between experiments and do not correlate with expected protein expression patterns.

💡 5 causes ✓ 5 fixes
Western Blot (Unexpected Multiple Bands) moderate

Altered Band Pattern from Excessive Cell Passage

Band patterns differ from expected or previously observed results when using cell lines that have been maintained in culture for extended periods. Multiple unexpected bands or altered expression levels appear compared to earlier passages.

💡 4 causes ✓ 5 fixes
Western Blot (Unexpected Multiple Bands) moderate

Extra Bands at 2× or 3× Expected Molecular Weight

Additional bands appear at molecular weights that are integer multiples (2×, 3×, or higher) of the expected target protein size, indicating oligomeric forms. These higher molecular weight bands suggest incomplete protein denaturation.

💡 4 causes ✓ 4 fixes
Western Blot (Unexpected Multiple Bands) moderate

Non-Specific Signal from Insoluble Protein in Well or Dye Front

Strong signal appears at the top of the gel (in or near the loading well) or at the bottom (dye front), unrelated to the expected molecular weight of the target protein. This is especially common for very high molecular weight proteins (>250 kDa).

💡 4 causes ✓ 5 fixes