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)
Immunohistochemistry (No Staining) severe

Complete absence of IHC signal due to reagent degradation

No staining is observed in immunohistochemistry despite proper protocol execution. Previously validated antibodies or detection kits fail to produce any signal.

💡 4 causes ✓ 6 fixes
Immunohistochemistry (No Staining) moderate

Nuclear protein not detected due to insufficient permeabilization

No staining observed when targeting nuclear proteins. Antibody cannot access nuclear compartment despite proper antibody concentration and incubation time.

💡 3 causes ✓ 4 fixes
Immunohistochemistry (No Staining) moderate

FFPE tissue shows no staining from incomplete deparaffinization

Paraffin-embedded tissue sections show no immunostaining. Residual paraffin creates a physical barrier preventing antibody access to tissue antigens.

💡 3 causes ✓ 4 fixes
Immunohistochemistry (No Staining) moderate

Weak or absent signal from inadequate antibody concentration or incubation

No detectable staining despite proper protocol execution. Insufficient antibody-antigen binding due to suboptimal concentration or incubation conditions.

💡 4 causes ✓ 5 fixes
Immunohistochemistry (No Staining) severe

Antibody not validated for IHC application or tissue type

Complete absence of staining with antibody that works in other applications. The antibody may not recognize native protein conformation present in tissue sections.

💡 4 causes ✓ 5 fixes
Immunohistochemistry (No Staining) moderate

Primary-secondary antibody incompatibility preventing detection

No signal detected despite proper primary antibody binding. Secondary antibody fails to recognize or bind to the primary antibody, breaking the detection chain.

💡 4 causes ✓ 5 fixes
Immunohistochemistry (No Staining) moderate

Loss of membrane protein staining after permeabilization

No staining is detected when targeting membrane proteins in IHC. Signal is absent despite antibody validation, suggesting structural damage to membrane epitopes.

💡 3 causes ✓ 4 fixes
Immunohistochemistry (No Staining) severe

Target protein absent or below detection threshold in tissue

No staining observed in experimental tissue samples. The target protein may not be expressed in the tissue type or developmental stage being examined, or present at undetectable levels.

💡 4 causes ✓ 6 fixes
Immunohistochemistry (No Staining) severe

Formaldehyde fixation masked epitopes preventing antibody binding

No staining in formaldehyde-fixed tissues despite validated antibody. Fixation-induced protein crosslinking masks epitopes and prevents antibody recognition.

💡 4 causes ✓ 5 fixes