Library · Biology & lab methods
What Western Blotting & Immunoblotting got wrong, from 45 theses
Summary written by a model from the records below: Western blotting and immunoblotting protocols frequently encounter failures stemming from antibody limitations, low protein abundance, and sample preparation artifacts. Researchers often abandon the method for alternatives like mass spectrometry or ELISA when targets fall below detection thresholds, antibodies cross-react non-specifically, or electrophoretic conditions disrupt critical epitopes. These records come from graduate theses, 2021 to 2026, each labeled with its degree where the repository states it, and 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.
Plus 10 problems the authors left open, listed at the end.
Failure modes, by theses reporting them
Select a mode to jump to its records.
Target protein abundance falls below the detection threshold of Western blotting
Endogenous target proteins present in low amounts, minute sample volumes, or complex matrices regularly fail to yield detectable signals on Western blots. In several investigations, the method lacked sufficient sensitivity to capture early expression events or faint bands, prompting researchers to pivot to techniques such as ELISA or mass spectrometry.
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 · PhD
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 · Tübingen · PhD
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 · Master's
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 · Doctorate
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 · University of Trento · PhD
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
An Analysis of How Lead Induces Changes in Histone Modification Levels that Impact Alzheimer’s Disease Gene Expression · Harvard · Master's
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 · PhD
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 · PhD
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.
Spatiotemporal host and viral transcriptomics, host restriction and DNA sensing after HSV-1 infection · Imperial · PhD
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 · PhD
Considered and rejected
Considered and rejected: Probing for ELK1 interaction in pulldown Western blots due to weak basal expression in RC nuclear extracts.
Determining transcription factor interactions with the add domain of DNA methyltransferases DNMT3A and DNMT3B · Boston University · Master's
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 · PhD
Considered and rejected
Considered and rejected: Continuing Western blot protein analysis of COX-2 in HMEC-1 cells was abandoned/redirected to HUVECs due to low protein yields and equipment/funding limitations.
THE EFFECTS OF CYCLOOXYGENASE-2 DURING COLORADO TICK FEVER INFECTION IN HUMAN ENDOTHELIAL CELLS · Sam Houston State University · Master's
Antibodies fail to recognize target antigens or bind denatured conformational epitopes
Primary antibodies often lack the affinity or functional capability to detect their intended targets in cell lysates, even when mRNA or recombinant controls are present. Furthermore, denaturing SDS-PAGE conditions or membrane immobilization disrupt conformational epitopes, preventing antibodies from binding native protein structures or specific phosphorylation states.
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 · PhD
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 · Master's
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 · PhD
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 · Master's
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 · PhD
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 · PhD
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
The Glycosyltransferase alpha-1,3-mannosyltransferase (ALG3) is an AKT Substrate that Regulates Protein N-glycosylation in Breast Cancer Cells · Harvard · PhD
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 · PhD
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 · PhD
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 · PhD
Tried and failed
immunoprecipitation with pan-phospho-serine/threonine western blotting applied to transcription factor phosphorylation detection. Outcome: no signal. Reason: pan-phospho antibodies showed no detectable phosphorylation change following signaling modulation
KLF6 Mediates de novo Ornithine Synthesis and Polyamine Production in Pancreatic Ductal Adenocarcinoma · Harvard · Master's
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 · Johns Hopkins · Master's
Non-specific antibody binding and cross-reactivity generate confounding bands
Commercial and polyclonal antibodies frequently produce intense non-specific bands, high background noise, and cross-reactivity across homologous proteins. These spurious signals persist in knockout or knockdown controls and corrupt standard loading controls like GAPDH, preventing reliable protein validation.
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
The modulation of NREM sleep by corticotropin-releasing hormone through the thalamic reticular nucleus · EPFL · PhD
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 · PhD
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 · PhD
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
The role of bla C̳T̳X̳-̳M̳ and porins in mediating ertapenem resistance among carbapenem resistant enterobacterales · UT Austin · PhD
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) · Nottingham · PhD
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 · University of Nevada, Reno · PhD
Considered and rejected
Considered and rejected: Rejected relying on CRHR1 antibodies for immunohistochemistry or Western blot quantification due to pervasive non-specific binding.
The modulation of NREM sleep by corticotropin-releasing hormone through the thalamic reticular nucleus · EPFL · PhD
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
Hydrocortisone, ascorbic acid, and thiamine therapy reduce sepsis-induced acute kidney injury in mice · Boston University · Master's
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 · Tübingen · PhD
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 · University College Dublin · PhD
Sample preparation issues, matrix interference, and transfer protocols compromise detection
Complex biological matrices like excess phospholipids compete with target proteins for membrane binding sites, while post-sacrifice degradation and small protein size impede standard detection. Additional technical bottlenecks arise from inadequate semi-dry transfer timing, loss of signal following membrane stripping, and shared epitope tags between co-expressed constructs.
Tried and failed
Epitope tag detection via western blotting applied to co-expressed recombinant proteins sharing identical tags. Reason: The overexpressed construct contained the same tag as the target protein, preventing specific detection
Role of an RNA granule in the jus-dependent mechanism of epileptogenesis in Drosophila · Cornell
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 · Tübingen · PhD
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.
The role of cyclic nucleotide-binding efflux pumps in Mycobacterium tuberculosis and antimicrobial resistance · Imperial · PhD
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 · PhD
Tried and failed
Western blot and immunostaining with commercial antibodies applied to short-lived transcription factors in animal tissue. Outcome: no signal. Reason: rapid protein degradation occurring post-sacrifice before detection
Defining the Pathogenic Mechanisms of Calcific Aortic Valve Disease through Single-Cell RNA Sequencing · Georgia Tech · PhD
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
IDENTIFICATION AND CHARACTERIZATION OF AFRICAN SWINE FEVER VIRUS PROTEINS AND THEIR CONTRIBUTION TO PROTECTION · Cornell · PhD
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 · Boston University
Technical irreproducibility and incomplete protein dissociation hinder quantification
Assays frequently suffer from excessive technical variability across replicates, leaving sample batches unusable for quantitative analysis. Furthermore, stable protein complexes such as streptavidin tetramers fail to fully dissociate and cause gel streaking, while other assays lack the electrophoretic resolution needed to resolve specific excision or lipidation bands.
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 · Queen's University · Master's
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 · PhD
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 · PhD
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
Evaluation of Cre Recombinase Models for the Study of Dnmt3a Mutations and Myeloid Bias in Clonal Hematopoiesis · Harvard · Master's
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 · Master's
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).
Characterizing the role of Staphylococcus aureus V8 protease in skin barrier damage and neuroimmune responses during host-pathogen interaction · Harvard · Master's
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).
Effects of Nicotianamine-biofortified wheat (Thriticum) on iron bioavailability, and physiological status, in vivo (Gallus gallus) · Cornell · Master's
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 · Saskatchewan · Master's
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 · PhD
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 · PhD
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.
Converting Conventional Antibodies into ScFv and ScFv-Fc Fusion Fragments to Improve Expression and Function · Harvard · Master's
Left open
Measure the expression and translation of identified DVG sequences into truncated proteins using western blotting. Blocker: Requires wet-lab facilities, cell cultures, plasmids, and reagents for western blot analysis.
Aberrant RNA replication of highly pathogenic avian influenza viruses and its impact on the mammalian-associated cytokine storm · Imperial · PhD
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.
Characterising embryonic stem cell-derived tenocytes and determining the changing role of scleraxis during tendon development · Cambridge · PhD
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 · PhD
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 · PhD
Who has worked on this
Authors of the public theses cited on this page, most-cited first.
- Leena Khelifa · Imperial · PhD · 2025
Development of a lateral flow device for neuroblastoma diagnosis - Loredana Cumpana · EPFL · PhD · 2024
The modulation of NREM sleep by corticotropin-releasing hormone through the thalamic reticular nucleus - Dawei Guo · Cambridge · PhD · 2022
On the mechanisms of substrate translocation by the ABC transporter MsbA - Emma Roberts · Imperial · PhD · 2022
The spatiotemporal control of CDK activity - Bailey Hannah · Nottingham · PhD · 2026
MicroRNA and Nrf2 pathway manipulation in human astrocytes: towards a therapeutic approach for Amyotrophic Lateral Sclerosis (ALS) - Max Peter Neukum · Tübingen · PhD · 2026
The influence of two PAX7 splice variants on WNT signaling activity during embryonic development in the chicken midbrain - Marie Andersen · Cornell · Master's · 2026
Effects of Nicotianamine-biofortified wheat (Thriticum) on iron bioavailability, and physiological status, in vivo (Gallus gallus) - Tobias William Plowman · Imperial · PhD · 2025
Retrotransposable element co-option in the evolution of immune networks - Sean Michael Watson · Harvard · PhD · 2025
Discovering conformation selective anti-EGFR nanobodies - Qifan Wen · Johns Hopkins · Master's · 2025
Decoding SENP Enzymes: Pivotal Regulators in SUMOylation Dynamics - Jessica Caroline Gomes Noll · Cornell · PhD · 2024
IDENTIFICATION AND CHARACTERIZATION OF AFRICAN SWINE FEVER VIRUS PROTEINS AND THEIR CONTRIBUTION TO PROTECTION - Jack Hassard · Imperial · PhD · 2024
Glycobiology studies of Influenza-A and SARS-CoV-2 respiratory viruses: the use of glycans as receptors and the role of host cells in viral protein glycosylation
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