Failure Case Library

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

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

Overexposed / signal saturation

Bands look thick and bloated, edges spill out, strong-signal differences are masked; not suitable for densitometry quantification because the linear range is lost.

💡 3 causes ✓ 4 fixes
Western Blot moderate

White bands / inverted signal (over-detection)

White (hollow) bands appear against a black background, or strong positive positions appear white instead of black — signal is so saturated that it appears inverted.

💡 3 causes ✓ 4 fixes
Western Blot moderate

Local blank patches / air bubble imprints

Certain regions of the membrane show no signal at all, forming round or irregular blank zones; often due to transfer failure in those areas while surrounding bands look normal.

💡 4 causes ✓ 4 fixes
Western Blot moderate

Uneven transfer / splotchy band pattern

Within a single band the intensity is uneven (light/dark patches); bands across different lanes have inconsistent shapes, with a splotchy appearance.

💡 4 causes ✓ 4 fixes
Western Blot severe

Band smearing / streaking

Bands have blurred edges, show vertical smearing or the entire lane is hazy; background is fogged, making band intensity comparisons unreliable.

💡 4 causes ✓ 4 fixes
Western Blot moderate

Weak bands (faint signal)

Bands are visible but barely detectable; long exposure times are needed, and the internal control also appears weak.

💡 5 causes ✓ 5 fixes
Western Blot severe

Multiple / non-specific bands

Bands appear at molecular weights that do not match the expected target; multiple bands present where only one is expected.

💡 5 causes ✓ 5 fixes
Western Blot critical

No bands at all (blank membrane)

Membrane is completely blank — neither target protein nor housekeeping control shows any signal after ECL exposure.

💡 5 causes ✓ 5 fixes
Western Blot minor

Smiling bands (curved bands)

Bands curve upward at the edges of the gel, appearing like a smile — the middle of the lane runs faster than the sides.

💡 4 causes ✓ 4 fixes