Chapter Four · failure evidence
What Western Blotting & Immunoblotting got wrong, from 56 dissertations
Western blotting and immunoblotting workflows frequently fail due to antibody cross-reactivity, insufficient assay sensitivity for low-abundance proteins, and denaturation-induced loss of conformational epitopes. Researchers also encounter severe reproducibility and detection hurdles stemming from membrane transfer problems, stripping failures, and unresolvable protein complexes. These records come from PhD theses at 20 institutions, 2021 to 2026. Each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Target protein abundance falls below the detection limit of the assay
Target proteins present at low endogenous levels or in minute sample volumes consistently fell below the detection threshold of standard immunoblotting assays. These detection limits frequently forced researchers to abandon Western blotting in favor of more sensitive alternatives like mass spectrometry, ELISA, or RT-qPCR.
Considered and rejected
Considered and rejected: Rejected western blotting as a clinical validation/detection method due to poor transfer reliability and low protein abundance in blood.
Development of a lateral flow device for neuroblastoma diagnosis · Imperial
Considered and rejected
Considered and rejected: Western blotting rejected for Parkin PBMC quantification due to insufficient sensitivity and weak band visualization at ~50 kDa
Untersuchung des Einflusses des Parkinson-assoziierten Proteins Parkin auf die Pathogenese der Spinozerebellären Ataxie Typ 3 · Publikationssystem UB Tuebingen
Tried and failed
immunoblotting with commercial polyclonal antibody applied to endogenous transmembrane protein detection. Outcome: no signal. Reason: antibody lacked sufficient sensitivity to detect low endogenous protein levels in whole cell lysate
Role of novel transmembrane protein 128 (TMEM128) in the regulation of NOX2 expression and ER Stress · Imperial
Considered and rejected
Considered and rejected: Rejected direct quantitative immunoblotting for untagged CheS due to poor antibody sensitivity; used mass spectrometry of 3xFLAG-CheS instead
Quantification and functional characterization of Sinorhizobium meliloti chemotaxis proteins · Virginia Tech
Considered and rejected
Considered and rejected: Rejected standard SDS-PAGE and western blotting for miniaturized sucrose gradient fractions from microfluidic chambers due to low protein yields, selecting dot blot analysis instead.
Commonalities between SMA and ALS: investigation of ribosome heterogeneity and translational defects · IRIS - UNITN - prod
Tried and failed
western blot protein detection applied to differentiated neuronal cell lysates. Outcome: no signal. Reason: target protein was below the limit of detection or antibody lacked sufficient binding affinity
Tried and failed
western blot for detecting targeted exon excision applied to conditional gene knockout validation. Outcome: no signal. Reason: insufficient signal or resolution to detect endogenous protein excision
Tried and failed
western blotting applied to low-abundance serum biomarker detection. Outcome: no signal. Reason: low target abundance and complex biological matrix interference prevented reliable protein transfer and detection
Development of a lateral flow device for neuroblastoma diagnosis · Imperial
Tried and failed
western blotting of endogenously tagged protein applied to whole organism protein lysates. Outcome: no signal. Reason: Endogenous protein expression levels were likely below the detection threshold in whole organism lysates
SMOC-1: THE LON AND THE SMA OF IT · Cornell
Considered and rejected
Considered and rejected: Western blotting rejected for evaluating low-MOI early-time-point IE expression due to signal falling below limit of detection.
Tried and failed
Western blot protein detection applied to cell-free expression products. Outcome: no signal. Reason: protein expression yield was below the detection limit of the assay
Resolving Signal Transduction in Complex Biological Environments · MIT
Tried and failed
affinity purification and Western blot quantification applied to recombinant proteins in bacteriophage lysates. Outcome: no signal. Reason: recombinant protein concentration levels in lysates were below detectable limits
NEW WAVE APPROACHES TO FOOD SAFETY: MULTI-HURDLE BIOCONTROL TECHNIQUES · Cornell
Lost to a baseline
In-cell Western assay required millimolar (mM) concentrations of TRAIL for detection despite nanomolar (nM) sensitivity in standard cell culture assays.
Considered and rejected
Considered and rejected: Rejected measuring biglycan and decorin protein levels in individual neonatal tendons via ELISA or Western blot due to low sensitivity and small tissue mass, relying instead on RT-qPCR mRNA expression.
Primary antibodies fail to detect their intended targets in biological extracts
Target-specific and tag-specific antibodies repeatedly failed to generate detectable bands despite confirmed transcription or expression of the target proteins. The lack of reliable or sufficiently high-affinity antibodies prevented protein detection in whole-cell lysates and complex tissue extracts.
Tried and failed
Western blot using target-specific polyclonal antibodies applied to yeast Dnt1 protein detection. Outcome: no signal. Reason: Antibodies failed to detect a specific target band on Western blots
The spatiotemporal control of CDK activity · Imperial
Tried and failed
polyclonal antibody western blot detection applied to specific protein isoform in whole lysates. Outcome: no signal. Reason: antibody failed to detect the target protein isoform in mixed embryo extracts
The Pro-Apoptotic Function of CED-9 Requires a CED-9-CED-4 Interaction · MIT
Tried and failed
Western blot protein quantification and validation applied to target protein in human tissue samples. Outcome: no signal. Reason: Lack of a specific and reliable antibody for target protein detection
The Association of Steroid 5-Alpha Reductase Type 2 and Estrogen Receptors in the Prostate · Harvard
Tried and failed
epitope-tagged protein detection via western blot applied to embryo cell lysates. Outcome: no signal. Reason: antibodies failed to produce detectable signal from tagged target protein in lysates
The discovery of novel proteins involved in cell polarity and the PAR complex in c. elegans · UT Austin
Tried and failed
Polyclonal antibody detection of conserved protein domains applied to Native bacterial effector proteins in Western blot. Outcome: no signal. Reason: Antibodies raised against conserved domains failed to detect native protein expression in bacterial strains
Hostile takeover factors of Pseudomonas · Imperial
Tried and failed
immunoblotting with custom polyclonal antibodies applied to target protein detection in whole-cell lysates. Outcome: no signal. Reason: antibodies lacked sufficient affinity or specificity to detect target directly without prior immunoprecipitation enrichment
Tried and failed
Western blot profiling of protein extraction fractions applied to post-translational modification detection in disease tissue. Outcome: no signal. Reason: Failed to detect specific modified species in tissue fractions despite positive immunohistochemistry staining
Capturing the heterogeneity of alpha-synuclein pathology in synucleinopathies · EPFL
Considered and rejected
Considered and rejected: Abandoned western blotting for KIM-1 after adjustments failed to remove high background or yield bands; switched to ELISA
Considered and rejected
Considered and rejected: Whole cell lysate western blotting using anti-His antibody-HRP failed to detect Rv3239c and Rv3728, prompting the switch to isolated membrane extractions.
Considered and rejected
Considered and rejected: Replaced western blotting of CerS1 with a commercial sandwich ELISA due to weak antibody staining with recombinant CerS1 protein
Novel therapeutics for treating pancreatic cancer · Imperial
Tried and failed
Western blot protein detection applied to target protein in neonatal primary cells. Outcome: no signal. Reason: protein expression was undetected despite presence of corresponding mRNA transcripts
Macrophages and T regulatory cells: immune mediators of allogeneic and xenogeneic Sertoli cells · Texas Tech
Considered and rejected
Considered and rejected: Western blot verification of SENP2 protein depletion was rejected due to the lack of a functional SENP2 antibody, using coverslip-based qRT-PCR and GFP-SENP2 reporters instead
Decoding SENP Enzymes: Pivotal Regulators in SUMOylation Dynamics · JScholarship
Antibodies exhibit non-specific binding and cross-reactivity with non-target proteins
Antibodies frequently exhibited off-target cross-reactivity and generated prominent non-specific bands in both control and knockout samples. High sequence homology among target proteins and non-specific binding by loading control antibodies further obscured valid signal interpretation.
Tried and failed
western blot validation of protein knockdown applied to homozygous knockdown mutant lines. Reason: antibody cross-reactivity produced an intense non-target band despite transcript depletion
Tried and failed
immunohistochemistry and western blotting with commercial antibodies applied to G-protein coupled receptor protein detection. Outcome: no signal. Reason: antibodies exhibited non-specific labeling across low-expression brain regions and produced non-specific western blot bands
Tried and failed
commercial antibody western blot detection applied to target protein in mouse tissue lysates. Outcome: no signal. Reason: antibodies lacked specificity for the target protein in mouse samples
The role of EROS in T cell biology · Imperial
Tried and failed
immunoblotting with commercial antibodies applied to gene knockout clone validation. Outcome: no signal. Reason: antibodies yielded no visible target bands or exhibited non-specific binding
Tried and failed
commercial antibody western blotting and co-immunoprecipitation applied to receptor-associated kinase interaction detection. Outcome: no signal. Reason: antibodies lacked specificity on western blot and failed to enrich target protein above background in lysates
Retrotransposable element co-option in the evolution of immune networks · Imperial
Tried and failed
multiplexed immunoblotting with primary antibodies applied to homologous bacterial outer membrane proteins. Reason: strong antibody cross-reactivity between homologous targets produced confounding signals despite normalization
Considered and rejected
Considered and rejected: Rejected Western blotting and immunocytochemistry for reliably quantifying Nrf2/HO-1 expression differences due to non-specific banding and major morphological confounding
MicroRNA and Nrf2 pathway manipulation in human astrocytes: towards a therapeutic approach for Amyotrophic Lateral Sclerosis (ALS) · University of Nottingham Repository
Considered and rejected
Considered and rejected: Rejected using 1D Western blotting for mouse in vivo SLIT2 cleavage assays due to an intractable non-specific ~50 kDa band detected by anti-SLIT2-C in Slit2-/- tissue, requiring replacement with 2D gel electrophoresis.
Balancing growth and repulsion: Slit proteolysis generates diverse signals in axon guidance · unevada
Considered and rejected
Considered and rejected: Rejected relying on CRHR1 antibodies for immunohistochemistry or Western blot quantification due to pervasive non-specific binding.
Considered and rejected
Considered and rejected: Abandoned GAPDH antibody as a Western blot loading control after it produced multiple non-specific bands, switching instead to Revert 700 Total Protein Stain.
The influence of two PAX7 splice variants on WNT signaling activity during embryonic development in the chicken midbrain · Publikationssystem UB Tuebingen
Considered and rejected
Considered and rejected: Halted further experiments using the N2a neuroblastoma cell line because Western blotting and RT-PCR demonstrated little to no UT-B expression and aberrant antibody binding at 40, 45, and 50 kDa.
Urea transporters in the mammalian brain · Research Repository UCD
Membrane transfer, stripping, and technical artifacts undermine assay reproducibility
Blotting performance was compromised by physical membrane issues including incomplete semi-dry transfer, loss of signal after membrane stripping, and lipid interference with protein adsorption. In addition, defective loading controls and high variability between technical replicates prevented reliable protein quantification.
Lost to a baseline
One wildtype atrial CaV1.2 sample excluded from western blot analysis due to a defective loading control
Considered and rejected
Considered and rejected: Separate Western blot membranes utilized for CHK2 detection because the antibody failed to bind after membrane stripping.
Molekulare Analyse der Rolle von SLy1 im p53-Signalweg · Publikationssystem UB Tuebingen
Considered and rejected
Considered and rejected: Abandoned western blot analysis for measuring collagen levels in ISMC lysates/supernatants due to inconsistent blot outcomes, switching to Sircol red assay.
Regulation of the intestinal smooth muscle cell phenotype by the antifibrotic drug nintedanib · Queens University Institutional Repository
Considered and rejected
Considered and rejected: Discontinued Bit61-pS132 antibody for Western blot quantification due to inconsistent technical replicates.
The spatiotemporal control of CDK activity · Imperial
Tried and failed
nitrocellulose membrane dot immunoblotting applied to protein quantification in lipid mixtures. Outcome: no signal. Reason: excess phospholipids competed with the target protein for membrane adsorption sites
On the mechanisms of substrate translocation by the ABC transporter MsbA · Cambridge
Tried and failed
quantitative Western blotting of extracted cell lysates applied to lactate-treated parasite protein samples. Outcome: data insufficient. Reason: replicate samples were unusable for quantitative analysis
Histone Lactylation Dynamics in Plasmodium falciparum · Harvard
Considered and rejected
Considered and rejected: 30-minute semi-dry transfer for MINAR1 Western blotting rejected in favor of 18-hour wet transfer due to non-specific banding and poor detection.
Molecular mechanisms of cardiovascular complications in kidney disease · OpenBU
Denaturing and reducing conditions disrupt epitope structures and protein complexes
Denaturing and reducing conditions inherent to standard SDS-PAGE disrupted conformational epitopes necessary for antibody binding and disassembled non-covalent complexes. Furthermore, stable multimeric proteins failed to resolve into discrete monomeric bands, and membrane immobilization in far Western assays masked critical binding interfaces.
Tried and failed
monoclonal antibody detection via western blotting applied to target protein in cell lysates. Outcome: no signal. Reason: specific antibody clones failed to bind denatured antigen in western blot assays
Novel Agonist TREM2 Monoclonal Antibodies Activate Syk-Associated Cell Signaling Pathways · Harvard
Tried and failed
Western blot antigen identification applied to conformational epitope detection. Outcome: no signal. Reason: The target epitope was conformational and disrupted by the denaturing conditions of the assay
Identification of meningococcal vaccine antigens using Reverse Vaccinology 2.0 · Imperial
Considered and rejected
Considered and rejected: Rejected SDS-PAGE and Western blotting for quantitative streptavidin determination because streptavidin tetramers are excessively stable and fail to fully dissociate into uniform monomeric bands, leaving streaks across gels.
On the mechanisms of substrate translocation by the ABC transporter MsbA · Cambridge
Considered and rejected
Considered and rejected: Rejected interpreting the 25 kDa band as non-covalent Id1 homodimerization, because SDS-PAGE western blots were run under reducing/denaturing conditions
INCREASED ID1 EXPRESSION IN ALCOHOLIC HEPATITIS AFFLICTED LIVERS · JScholarship
Tried and failed
reciprocal far Western blotting applied to detecting protein-protein interaction. Outcome: no signal. Reason: immobilization of the bait protein on the membrane likely masked the binding interface or caused denaturation
The role of FACT in nucleosome inheritance · Imperial
Tried and failed
western blot protein detection applied to small recombinant viral proteins. Outcome: no signal. Reason: Small protein size likely prevented successful detection via standard Western Blot despite positive immunofluorescence
Left open by the authors
Problems the authors named and did not get to.
Left open
Confirm protein-level tight junction cleavage (claudin-1, occludin, ZO-1, desmoglein-1) using western blot or immunostaining. Blocker: Requires a biological wet lab, cell cultures/tissues, antibodies, and experimental wet-lab assays (western blot, immunostaining).
Left open
Perform Western blotting on harvested Gallus gallus intestinal and liver tissues to measure transporter protein abundance and functional activity. Blocker: Requires a wet lab, animal tissue samples, antibodies, and physical assay apparatus (Western blot equipment).
Left open
Repeat western blot experiments on rat tissue samples using β-actin as the loading control instead of total protein normalization. Blocker: Requires wet lab facilities, tissue samples, and physical reagents to perform Western blotting.
THE EFFECTS OF ORAL ARGININE ON ITS METABOLIC PATHWAYS IN SPRAGUE-DAWLEY RATS · HARVEST
Left open
Validate proteomics findings using western blot or cell-based experimental assays. Blocker: Requires a wet biology laboratory, biological reagents, and experimental assays (western blot).
Neutrophil biomechanical properties and immune function in health and inflammatory disease · Cambridge
Left open
Generate knockout mutants using a kanamycin resistance cassette to confirm the identity of the 63 kDa antigen via western blot comparisons. Blocker: Requires wet-lab facilities, bacterial cultures, and biochemical assays (western blot, mutagenesis).
Identification of pneumococcal vaccine antigens using Reverse Vaccinology 2.0 · Imperial
Left open
Perform non-denaturing Western blots on trastuzumab and anti-ApoE4 constructs to assess antibody function in native conformation. Blocker: Requires a wet lab, biological reagents, expressed antibody constructs, and Western blotting equipment.
Left open
Perform quantitative Western blotting to measure scleraxis knockdown and protein expression of TNMD, Collagen 1, Collagen XIV, and RANKL. Blocker: Requires a wet lab, antibodies, cell culture systems, and biological samples.
Left open
Perform antiphosphotyrosine or phospho-ERK Western blots to evaluate agonism and antagonism of selected anti-EGFR nanobodies in functional cell assays. Blocker: Requires wet lab cell culture and Western blot experimental apparatus with the physical nanobody samples
Discovering conformation selective anti-EGFR nanobodies · Harvard
Left open
Test GDF15 in the NRTN bioassay and vice versa to assess specificity, measuring phosphorylated ERK and AKT via Western blot. Blocker: Requires wet-lab biological facilities, recombinant proteins, cell bioassays, and Western blotting reagents.
Characterisation of Prodomains in the GDNF Family · Cambridge
Left open
Perform a Western blot on harvested HeLa plasmid cell lines RBM6-1, RBM6-3, and RBM6-4 to verify expression. Blocker: Requires wet-lab equipment, reagents, and the specific harvested HeLa plasmid cell lines.
Exploring RBM6 Overexpression: Impact on Cell Division and Gene Splicing in HeLa Cells · UT Austin
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