Library · Biology & lab methods
What Gel Electrophoresis & Blot Transfer got wrong, from 28 theses
Summary written by a model from the records below: Electrophoretic separation and blotting techniques frequently encounter technical bottlenecks related to antibody quality, target abundance, sample stability, and physical transfer constraints. Across diverse experimental settings, researchers often abandon these assays or switch to alternative methods when probes lack specificity, starting material is insufficient, samples degrade, or transfer apparatuses fail. 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.
Inadequate antibody specificity and binding affinity during immunoblotting
Immunoblotting procedures frequently failed because primary antibodies lacked the required sensitivity, affinity, or specificity to detect target antigens above background noise. Several antibody clones failed to bind denatured proteins on membranes or yielded high false-positive rates and non-specific binding.
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
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
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
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
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
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
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: 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
Considered and rejected
Considered and rejected: Discarded exclusive reliance on single-protein commercial line blots due to high false-positivity rates compared to tissue indirect immunofluorescence assay screening.
Characterising Autoimmune Neurologic Disorders: Biomarker Identification and Clinical Phenotyping · University College Dublin · PhD
Considered and rejected
Considered and rejected: Abandoned western blotting for ICAM-1 and VCAM-1 detection in hPAECs due to antibody failure, replacing the method with flow cytometry.
The iron regulatory axis of pulmonary artery vascular cells; disruption and implications for PAH subtypes · Imperial · Doctorate
Low target abundance and excessive sample input requirements
Target proteins and nucleic acids frequently fell below the detection threshold in whole cell lysates, serum, and small tissue specimens. Furthermore, assays like Southern and Western blotting were rejected because slow-growing cultures and limited biological samples could not satisfy the high starting material demands.
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 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
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
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: 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
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
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
Considered and rejected
Considered and rejected: Rejected Southern blot and flow-FISH for telomere length in large-scale studies in favor of qPCR due to DNA input requirements and labor constraints
Assessing relationships between discrimination and health: emphasis on measurement and methodology · Harvard · PhD
Considered and rejected
Considered and rejected: 2-D gel electrophoresis coupled with Southern blotting to visualize replication intermediates was rejected because it requires a large quantity of starting material unattainable from slow-growing SI colonies
Investigation of the Evolution and Escape Process of het-6 Non-Self Recognition in Neurospora crassa · Carleton University · PhD
Tried and failed
Northern blotting for low-abundance non-coding RNA applied to tRNA detection across growth conditions. Outcome: no signal. Reason: Expression levels were below the detection threshold across almost all tested growth conditions and species
The Evolution of Alternative Genetic Codes · Harvard · PhD
Sample degradation, aggregation, and incomplete denaturation causing abnormal gel migration
Rapid degradation of labile transcripts and proteins created diffuse smearing and non-specific fragments that obscured target bands on electrophoresis gels. In addition, hydrophobic protein aggregation and hyper-stable protein multimers failed to enter resolving gels or separate into clean monomeric bands.
Tried and failed
Southern blot cleavage assay applied to plasmid DNA cleavage detection. Outcome: no signal. Reason: Non-specific degradation products appeared in negative control plasmids even without tissue extracts, obscuring specific cleavage.
Increasing age alters Z-DNA-induced genetic instability · UT Austin · PhD
Considered and rejected
Considered and rejected: Rejected Western blot protein screening for cell death toxin screens due to protein degradation; switched to IVTT-expressed DNA barcodes.
Microdroplet assay development for metabolic engineering and synthetic biology applications · UT Austin · PhD
Tried and failed
preclearing, extended denaturation, reducing agents, and nuclease applied to RNA pull-down western blot lysates. Reason: failed to eliminate high-molecular-weight non-specific smearing
Molecular mechanisms for the post-transcriptional regulation of tissue factor expression · Imperial · PhD
Considered and rejected
Considered and rejected: Rejected Northern blotting for quantifying poly(A) site mutation effects due to insufficient resolution.
Transcript cleavage and polyadenylation in plants · University of Washington · PhD
Tried and failed
native gradient PAGE applied to hydrophobic outer membrane proteins. Reason: hydrophobic membrane protein aggregated at gel top instead of entering resolving pores
The interaction of glycosylated major outer membrane protein of Campylobacter jejuni with natural and synthetic ligands · Nottingham · PhD
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
Transfer apparatus limitations and transfer inefficiencies for large proteins
Blotting protocols were compromised by technical transfer failures such as excessive heat build-up during semi-dry transfer and poor membrane detection. Researchers also encountered severe difficulties efficiently transferring very high-molecular-weight proteins from gels onto membranes.
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
Tried and failed
Western blot protein detection applied to high-molecular-weight proteins. Outcome: no signal. Reason: Technical difficulties transferring and detecting very large 350 kDa proteins
Uncovering the Role of Lamin A/C in Nuclear Mechanics and Regulation of Gene Expression in Health and Disease · Cornell · PhD
Considered and rejected
Considered and rejected: Semi-dry Western blot transfer was abandoned due to heat build-up and transfer failure, replaced with tank wet transfer
Structural analysis of APOLLO, a candidate apomixis factor · Saskatchewan · Master's
Left open by the authors
Problems the authors named and did not get to.
Left open
Confirm the enrichment of IL-1B on a protein level using ELISA or western blot. Blocker: Requires wet lab equipment, biological samples/mice, and wet lab assays (ELISA/western blot).
Exploring the Role of Interleukin 1 Beta (IL-1B) in Immune Checkpoint Inhibitor-induced Colitis · Imperial · Master's
Left open
Develop a high-throughput assay to measure endoplasmic reticulum stress to replace standard Western blotting. Blocker: Requires wet lab equipment, cell cultures, and biological assay development reagents
Phenotypic High-Throughput Screening to Identify Small-Molecule Inhibitors of Beta-Cell Glucolipotoxicity · Harvard · PhD
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
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
Analyse specified inflammatory markers and cytokines by ELISA and Western blot in PLA-stratified tissue samples. Blocker: Requires a wet lab, post-mortem tissue samples, and wet-lab assay equipment (ELISA/Western blot)
Tau-proximity ligation assay reveals extensive previously undetected pathology prior to neurofibrillary tangles in preclinical Alzheimer's disease · Imperial · PhD
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
Characterize CRISPR/Cas9 knock-in efficiency and integration accuracy using sequencing and Southern blot assays. Blocker: Requires wet lab facilities, biological samples, and wet experimental equipment for Southern blotting and sequencing
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
Quantify total TERRA RNA reduction following TERRA ASO transfection in cancer cells using Northern blot analysis. Blocker: Requires a wet-lab environment, cell culture facilities, and reagents for Northern blotting and ASO transfection
Telomeric localization of the TElomeric Repeat-containing RNA TERRA impairs telomerase activity in human cancer cells · University of Trento · PhD
Left open
Deliver anti-synuclein antibodies across the blood-brain barrier into a Parkinson's mouse model using transferrin-functionalized MC3 lipid nanoparticles. Blocker: Requires wet-lab lipid nanoparticle formulation, antibody bioconjugation, and in vivo Parkinson's mouse model experiments.
Development and application of bioconjugation-based platforms for intracellular protein delivery with lipid nanoparticles · Cornell · 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 - Michael Gilligan · University College Dublin · PhD · 2026
Characterising Autoimmune Neurologic Disorders: Biomarker Identification and Clinical Phenotyping - 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 - Azmain Alamgir · Cornell · PhD · 2025
Development and application of bioconjugation-based platforms for intracellular protein delivery with lipid nanoparticles - Ghazaleh Nourparvar · Carleton University · PhD · 2025
Investigation of the Evolution and Escape Process of het-6 Non-Self Recognition in Neurospora crassa - Elizabeth Culpepper · Harvard · Master's · 2024
Evaluation of Cre Recombinase Models for the Study of Dnmt3a Mutations and Myeloid Bias in Clonal Hematopoiesis - Adrija June Navarro · Harvard · PhD · 2024
The Glycosyltransferase alpha-1,3-mannosyltransferase (ALG3) is an AKT Substrate that Regulates Protein N-glycosylation in Breast Cancer Cells - Nolan T. Tucker · MIT · PhD · 2024
The Pro-Apoptotic Function of CED-9 Requires a CED-9-CED-4 Interaction - Courtney Chatterton-Bartley · Imperial · PhD · 2023
Molecular mechanisms for the post-transcriptional regulation of tissue factor expression - Maggie Zoe Patrikas · Harvard · Master's · 2023
An Analysis of How Lead Induces Changes in Histone Modification Levels that Impact Alzheimer’s Disease Gene Expression - Elisavet Velentza Almpani · Imperial · PhD · 2023
Tau-proximity ligation assay reveals extensive previously undetected pathology prior to neurofibrillary tangles in preclinical Alzheimer's disease
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