Library · Biology & lab methods
What In Vitro Binding Assays got wrong, from 43 theses
Summary written by a model from the records below: The evaluated records describe experimental difficulties and methodological bottlenecks encountered across diverse in vitro binding assays. Major obstacles include non-specific surface adsorption, poor correlation between in vitro measurements and cellular biology, sensor detection failures, and mathematical modeling breakdowns during kinetic analysis. These records come from graduate theses, 2021 to 2025, 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 9 problems the authors left open, listed at the end.
Failure modes, by theses reporting them
Select a mode to jump to its records.
Non-specific surface adhesion and matrix background obscure specific binding signals
Assays such as ELISA, immunoprecipitation, TR-FRET, and membrane transport frequently suffered from high background noise and false positives caused by non-specific sticking to plates, beads, and lipid membranes. Unwanted adsorption of probes, reporters, and hydrophobic compounds masked true target interactions or led researchers to abandon problematic surface coatings and resins.
Tried and failed
baculovirus particle ELISA using recommended coating dilution applied to antibody specificity screening. Reason: Recommended dilution caused false positive non-specific binding from direct antibody binding to the microplate
Specificity and other Biophysical Properties to Guide Developability of Agonistic Bispecific Antibodies · Harvard · Master's
Tried and failed
TR-FRET assay for protein-protein interactions applied to membrane-associated enzyme binding affinity. Reason: Full-length protein exhibited non-specific stickiness, yielding false-positive saturable binding curves with negative controls.
In vitro and in vivo studies of two essential MRSA cell wall synthesis enzymes · Harvard · PhD
Considered and rejected
Considered and rejected: Transwell bidirectional transport assay was rejected for assessing THC transport due to excessive intracellular retention and non-specific binding, prompting cellular accumulation assays
(-)-Δ9-tetrahydrocannabinol and pregnancy: transporter-mediated tissue distribution and drug interactions · University of Washington · PhD
Tried and failed
photoaffinity labeling with mass spectrometry target identification applied to small molecule protein target discovery. Outcome: no signal. Reason: candidate protein enrichment was a non-specific false positive, failing thermal shift validation
Identifying the modes and mechanisms of action of transmission-blocking antimalarials against Plasmodium falciparum · Imperial · PhD
Lost to a baseline
Hydrophobic long-chain alkanethiol SAM-functionalized Au surfaces exhibited significantly stronger non-selective binding (NSB) of BSA compared to polystyrene 96-well ELISA plates and hydrophilic dextran-functionalized CM5 chips
Methods to Improve the Stability and Selectivity of Electronic Biosensors · Georgia Tech · PhD
Considered and rejected
Considered and rejected: Rejected non-fixed live-cell staining in MRI phantoms because absence of serum blocking caused extensive non-specific binding of SPIONs.
Magnetic resonance strategies for tracking immune cells · Nottingham · PhD
Considered and rejected
Considered and rejected: Ceased testing synthetic peptide binders (RRGW, WGRR, Fc-III-4C) because binding signal was below or equivalent to non-specific background binding to ELISA plates.
Collagen Anchoring Agonist Antibodies for Cancer Immunotherapy · MIT · PhD
Considered and rejected
Considered and rejected: Rejected poly-L-lysine (PLL) slide coating due to prominent non-specific probe binding background
Single-cell imaging assay for rapid antimicrobial resistance testing · Oxford · Master's
Considered and rejected
Considered and rejected: SNAP-tag system for cuticle pore permeability assay due to non-specific background binding to the pharynx.
Patterning of the apical extracellular matrix at sense organs · Harvard · PhD
Tried and failed
liposome sedimentation assay for membrane protein recruitment applied to protein-protein complex recruitment to lipid membranes. Outcome: no signal. Reason: proteins exhibited high non-specific intrinsic binding to synthetic liposomes without target complex present
STRUCTURAL AND BIOCHEMICAL MECHANISMS OF THE ACTIVATION OF ARF1 AT THE GOLGI · Cornell · PhD
Tried and failed
intact protein enrichment before digestion applied to modified peptide identification in proteomics. Outcome: worse than baseline. Reason: non-specific binding and interference from unlabeled background peptides reduced target identification depth
Developing novel mass spectrometry-based approaches to globally investigate protein glycosylation · Georgia Tech · PhD
Tried and failed
co-immunoprecipitation of endogenous proteins applied to transcription factor protein-protein interactions. Outcome: no signal. Reason: antibody non-specific binding caused high background signal present in both control and knockout lysates
B-MYB and FOXM1 drive cell division through cooperative regulation of cell cycle genes. · Harvard · PhD
Tried and failed
metabolic inhibition to reduce background substrate binding applied to radiolabeled lipid uptake assays. Outcome: no signal. Reason: metabolic poisons halt active processing but do not eliminate passive, non-specific hydrophobic membrane adsorption
CHARACTERIZING THE ROLES FOR HOST-DERIVED LIPIDS DURING MYCOBACTERIUM TUBERCULOSIS INFECTION · Cornell · PhD
Tried and failed
whole-cell ELISA for monoclonal antibody screening applied to bacterial antigen-specific human monoclonal antibodies. Outcome: no signal. Reason: produced high non-specific background binding to negative controls and assay surfaces
Identification of pneumococcal vaccine antigens using Reverse Vaccinology 2.0 · Imperial · PhD
Tried and failed
high temperature thermal lysis of membrane vesicles applied to hydrogel microparticle biomolecule assays. Reason: high heat caused lipid and lipoprotein fouling leading to severe non-specific probe background binding
Extracellular Vesicle Capture and microRNA Detection · MIT · PhD
Tried and failed
single molecule magnetic bead capture assay applied to low concentration DNA detection. Outcome: no signal. Reason: severe non-specific binding of protein reporter to carboxylated magnetic beads caused high background
Applications of Engineered Proteins in Redox Biology and Biomarker Detection Assay Development · MIT · PhD
Considered and rejected
Considered and rejected: Rejected column-based online immunoprecipitation due to high background signals from unspecific binding and carryover
Biomarker qualification for drug-induced liver injury across species using immunoaffinity mass spectrometry · Tübingen · PhD
Considered and rejected
Considered and rejected: Rejected inside-out membrane vesicular transport assays due to high non-specific binding of lipophilic drugs and rapid leakage of high-permeability compounds.
Predicting Human Fetal Drug Exposure to Placental P-glycoprotein Substrates by PBPK Modeling and Simulation · University of Washington · PhD
Considered and rejected
Considered and rejected: Rejected 254 nm UVC crosslinking due to high background and poor specificity for RNA-binding motifs.
Investigation Of Rna-Protein Interactions In Prc2 Function · Penn · PhD
Tried and failed
fluorescence polarization competition assay for screening binding applied to macrocyclic protein-protein interaction inhibitors. Outcome: no signal. Reason: peptide precipitation and non-specific plate sticking caused elevated anisotropy and decreased total fluorescence artifacts
Generating macrocyclic inhibitors of protein-protein interactions · EPFL · PhD
In vitro binding affinities and motifs fail to predict cellular or in vivo activity
Recombinant binding metrics and sequence motifs identified in vitro frequently failed to correlate with genomic occupancy, tissue transcytosis, or functional cellular activation. Researchers noted that simplified cell-free assays lacked physiological context, missing regulatory partners and post-translational modifications necessary to reflect in vivo mechanisms.
Tried and failed
fixed tissue immunohistochemistry binding assay applied to predicting in vivo antibody transcytosis. Outcome: no signal. Reason: fixed-tissue binding affinity failed to capture dynamic cellular transport and transcytosis mechanisms in vivo
Development and Exploration of Immunohistochemistry as An in vitro Assay for Predicting Antibody Transcytosis in vivo · Harvard · Master's
Tried and failed
in vitro binding affinity profiling applied to predicting in vivo genomic occupancy. Outcome: did not generalise. Reason: in vitro specificity differences did not predict cellular binding site occupancy
Regulatory consequences of variation in transcription factor-DNA binding · Harvard · PhD
Tried and failed
fixed cell microarray screening for protein interactions applied to validating viral glycoprotein host receptors. Outcome: no signal. Reason: Microarray binding hits failed to reproduce when assayed on live cells via flow cytometry.
Structural basis of antibody-mediated immunity against Crimean-Congo hemorrhagic fever virus glycoproteins · UT Austin · PhD
Tried and failed
peptide screening using surface plasmon resonance affinity applied to T cell receptor functional activation prediction. Outcome: no signal. Reason: Micromolar binding affinity failed to translate into functional downstream cellular activation
Tried and failed
in vitro high-throughput motif enrichment screening applied to identifying in vivo protein-RNA binding motifs. Outcome: did not generalise. Reason: weakly enriched in vitro motifs failed to show corresponding binding peaks in in vivo crosslinking validation
Tried and failed
predicting in vivo occupancy from in vitro affinity applied to transcription factor binding domain mutants. Outcome: did not generalise. Reason: in vivo binding showed a sharp threshold loss rather than reflecting the continuous in vitro affinity gradient
Regulatory consequences of variation in transcription factor-DNA binding · Harvard · PhD
Tried and failed
in vitro high-throughput secondary motif identification applied to in vivo RNA-protein binding enrichment. Outcome: did not generalise. Reason: secondary low-affinity binding motifs detected in vitro lacked robust enrichment in in vivo crosslinking-immunoprecipitation peaks
Lost to a baseline
Branched peptides K(Ac)-Y-E-L-K(Ac)-a-a, K(Ac)-Y-E-L-K(H-Y-M)-a-a, and K(Ac)-Y-E-L-K(Y-E-T)-a-a failed to outperform lead dual-functionalized peptides in in vitro vancomycin antagonism at a 5:1 molar ratio despite reported up to 28-fold higher binding affinity.
The Development of Peptide-Based Alternative Approaches to Combat Antibiotic-Resistant Bacterial Infections · University of Nevada, Reno · Doctorate
Considered and rejected
Considered and rejected: Rejected exploratory in vitro binding assays for factor specificity, favoring in vivo cross-linking/perturbation due to lack of cooperative RBPs and post-translational modifications in vitro.
Quantitative modeling of 5' splice site subclass regulation and evolution · MIT · PhD
Considered and rejected
Considered and rejected: Rejected Thioflavin T (ThT) labeling in vitro for generating ground truth due to lack of cellular context, potential non-specific binding, and incompatibility with live dynamic intracellular interactions
Intelligent and Self-Driving Microscopy of Protein Aggregation in Neurodegenerative Diseases · EPFL · PhD
Optical and biophysical transducers fail to detect target binding interactions
Surface plasmon resonance, localized surface plasmon resonance, circular dichroism, and zeta potential assays often produced no detectable signal changes upon target incubation. Insufficient surface density, low refractive index contrast, steric hindrance, or pipeline instability resulted in false negatives despite functional inhibition or structural predictions.
Tried and failed
surface plasmon resonance validation screening applied to RNA-binding protein small molecule interactions. Outcome: unstable. Reason: could not establish robust and reproducible binding assay pipelines for the protein panel
High Throughput Screening for Small Molecule Interactions with Nucleic Acid Binding Proteins · MIT · PhD
Tried and failed
molecular docking virtual screening compared to biophysical assays applied to fragment library screening against RNA targets. Outcome: no signal. Reason: computational docking hits showed zero overlap with surface plasmon resonance experimental binding hits
Can In-Silico Computational Virtual Screening of Fragment Libraries Model In-Vitro Biophysical Screening Methods with Similar Results to Identify Novel RNA-Binding Chemical Matter · Harvard · Master's
Tried and failed
biophysical assays to detect direct target binding applied to enzyme inhibitor validation. Outcome: no signal. Reason: SPR and DSF failed to detect binding interaction despite functional inhibition
Targeting the streptococcal virulence factor SpyCEP as a novel therapeutic approach · Imperial · PhD
Tried and failed
responsive polymer shell coating for plasmonic biosensing applied to protein detection via LSPR shifts. Outcome: no signal. Reason: Target protein binding caused no concentration-dependent localized surface plasmon resonance wavelength shifts across various polymerizations
Impact of synthesis technique and compositon of polymer coatings for semiselective gold nanoshell-based biosensors · UT Austin · PhD
Tried and failed
circular dichroism to detect surface antibody coatings applied to protein nanoparticle surfaces. Outcome: no signal. Reason: antibody concentration on nanoparticle surfaces was below instrument detection limit despite functional surface binding
Engineering Biomaterial - Protein Corona Interactions to Mediate Biological Responses · Georgia Tech · PhD
Tried and failed
split-ubiquitin yeast two-hybrid assay applied to extracellular receptor-ligand interactions. Outcome: no signal. Reason: assay failed to detect extracellular peptide-receptor binding, producing false negatives despite high-confidence structural predictions
Tried and failed
direct protein physisorption onto sensor surface applied to optical biosensing transducers. Outcome: no signal. Reason: insufficient surface binding density or low refractive index contrast prevents measurable optical resonance shifts
All-Dielectric Nanophotonic via Glass Fluid Instabilities · EPFL · PhD
Solution biophysical assays fail under challenging analyte conditions or extreme concentrations
Methods including isothermal titration calorimetry and equilibrium dialysis broke down when evaluating weak-affinity interactions, insoluble compounds, or redox-active metals. Assays were impeded by protein self-association at high concentrations, low signal-to-noise ratios, precipitation, and interfering additives like residual solubilizing polymers.
Considered and rejected
Considered and rejected: Rejected surface plasmon resonance (SPR), ITC, and MST for determining ion binding affinity due to molecular weight mismatch or redox metal instability, proposing DSF instead
Copper A Novel Regulator Of Perk · Penn · PhD
Tried and failed
polymer-based membrane protein solubilisation applied to radioligand binding assays. Outcome: no signal. Reason: residual free polymer directly interfered with ligand binding, eliminating specific binding signal
Structural and functional study of vesicular acetylcholine transporter (VAChT) · Imperial · PhD
Considered and rejected
Considered and rejected: Rejected NMR titration and isothermal titration calorimetry (ITC) for measuring aqueous nanohoop-fullerene binding affinities due to low sensitivity (>10^6 M^-1) and precipitation artifacts.
Functionalized Carbon Nanohoops: Nitrogen-Doped Partial Belts, Macrocyclic Ligands, and The Inherent Strain That Affects Their Chemical Properties · University of Oregon · PhD
Considered and rejected
Considered and rejected: Traditional binding equilibrium and X-ray crystallographic approaches for mapping the TCA binding site were rejected due to the low-millimolar binding affinity
Modulation of eukaryotic topoisomerase II activity by naturally occurring small molecules · Johns Hopkins · PhD
Considered and rejected
Considered and rejected: Rejected using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) for the benzophenanthridinone class (968) due to high protein concentration requirements where GAC forms dimers/tetramers rather than binding monomers.
THE ALLOSTERIC REGULATION OF GLUTAMINASE ENZYMES: DRIVING GLUTAMINE CATABOLISM IN CANCER CELLS · Cornell · PhD
Theoretical and kinetic binding models fail to fit complex or multivalent experimental data
Standard mathematical models frequently failed to converge or match experimental titration curves generated by isothermal titration calorimetry and surface plasmon resonance. These fitting failures arose when models could not accommodate multi-site binding, multivalent interactions, surface saturation limits, or nucleic acid hybridization anomalies.
Tried and failed
surface plasmon resonance binding affinity modeling applied to fragment library screening on structured RNA. Outcome: did not converge. Reason: complex multi-site binding and solubility limits prevented reaching surface saturation needed for kinetic fitting
Can In-Silico Computational Virtual Screening of Fragment Libraries Model In-Vitro Biophysical Screening Methods with Similar Results to Identify Novel RNA-Binding Chemical Matter · Harvard · Master's
Tried and failed
predicting surface hybridization using thermodynamic binding models applied to nucleic acid microarray probe binding. Outcome: no signal. Reason: theoretical free energy and GC content did not correlate with observed surface hybridization intensity
Measuring the Effects of Viral RNA Structure on Capsid Self-Assembly · Harvard · PhD
Tried and failed
Sequential binding models for ITC data fitting applied to protein-RNA homodimer binding isotherm analysis. Outcome: did not converge. Reason: Total concentration assumptions and sequential binding models failed to capture complex or asymmetric multivalent binding equilibria
A structure-based mechanism for recognition of oskar mRNA by Tropomyosin 1 · Harvard · PhD
Lost to a baseline
Equilibrium multivalent binding theory failed to accurately fit the steepness of adsorption for the strongest binder (Delta G0 = -17 kBT), requiring an empirical two-parameter logistic fit instead.
Controlling and designing the binding properties of multivalent systems: a coarse-grained modelling and simulations approach · Imperial · PhD
Left open by the authors
Problems the authors named and did not get to.
Left open
Test whether a simple cell or protein binding assay predicts loss of binding and in vivo transcytosis activity of antibody mutants. Blocker: Requires wet lab experiments with cell or protein binding assays and mutant antibodies
Development and Exploration of Immunohistochemistry as An in vitro Assay for Predicting Antibody Transcytosis in vivo · Harvard · Master's
Left open
Perform in vitro binding assays between the RetS Dismed2 domain and mucin glycans to probe direct glycan-binding mechanism. Blocker: Requires a wet lab, recombinant protein purification, and in vitro glycan binding assays.
Left open
Test whether full-length BtszB or Btsz-CT binds G-actin and promotes actin assembly or disassembly in vitro. Blocker: Requires purified proteins and wet-lab in vitro actin polymerization/binding assays
Bitesize bundles F-actin and influences actin remodeling in syncytial Drosophila embryo development · MIT · PhD
Left open
Investigate specific secretion and internalization mechanisms of cell-surface RNA-binding proteins in neurodegenerative disease and cancer models. Blocker: Requires wet lab biological experiments, disease cell lines, and molecular assay apparatus.
Cell-Surface RNA-Binding Proteins · Harvard · PhD
Left open
Test in vitro whether Sld3/7 forms a functional heterotetramer and preferentially binds Mcm2-7 double hexamers. Blocker: Requires wet lab biochemical assays, protein purification, and binding analysis
Single-molecule studies of the mechanism of eukaryotic helicase activation · MIT · PhD
Left open
Perform functional validation of increased VDR binding and activation in APOE2-expressing microglia using TF ChIP-seq, knockout, or overexpression assays. Blocker: Requires a wet lab and biological experimental assays (TF ChIP-seq, KO, overexpression)
Cell type-specific genomic signatures, past and present: using mammalian data to gain insight into human brain disorders · Imperial · PhD
Left open
Perform high-throughput experimental testing and library binding assays to validate the in vitro activity of computationally designed peptide binders. Blocker: Requires a wet biology laboratory and experimental assay infrastructure
Computational methods for the structure-based design of protein-binding peptides · MIT · PhD
Left open
Validate binding affinity of ambiguous or fluorescence-quenching MST hit compounds against G9a using surface plasmon resonance assays. Blocker: Requires a wet lab, surface plasmon resonance instrumentation, purified recombinant G9a protein, and physical compound samples
Development and evaluation of potential co-factor competitive inhibitors for the histone lysine methyltransferase G9a · Imperial · PhD
Left open
Identify additional hits from antibody libraries and characterize full IgG binding affinity using surface plasmon resonance. Blocker: Requires wet lab facilities, surface plasmon resonance equipment, and physical antibody libraries/samples
ENGINEERING THE HUMORAL RESPONSE TO GENERATE ANTIGEN-SPECIFIC ANTIBODIES · Cornell · PhD
Who has worked on this
Authors of the public theses cited on this page, most-cited first.
- Ameya Dhatri Madduri · Harvard · Master's · 2023
Can In-Silico Computational Virtual Screening of Fragment Libraries Model In-Vitro Biophysical Screening Methods with Similar Results to Identify Novel RNA-Binding Chemical Matter for Treatment of Myo - Kian Hong Kock · Harvard · PhD · 2022
Regulatory consequences of variation in transcription factor-DNA binding - Jiao Qiao · Harvard · Master's · 2022
Development and Exploration of Immunohistochemistry as An in vitro Assay for Predicting Antibody Transcytosis in vivo - Bridget E. Begg · MIT · PhD · 2022
Concentration-dependent Splicing through Suboptimal Motifs Enables Waves of Gene Regulation in Neuronal Development - Connor Jens Kenny · MIT · PhD · 2025
Quantitative modeling of 5' splice site subclass regulation and evolution - Caroline A. Martin · MIT · PhD · 2025
Mapping signaling networks and rapidly evolving genes in the developing Arabidopsis seed at single-nucleus resolution - Jonathan Bryce Perr · Harvard · PhD · 2025
Cell-Surface RNA-Binding Proteins - Clare Canavan · Harvard · PhD · 2025
In vitro and in vivo studies of two essential MRSA cell wall synthesis enzymes - Teresse de Rham · Harvard · Master's · 2025
Specificity and other Biophysical Properties to Guide Developability of Agonistic Bispecific Antibodies - Xin Chen · University of Washington · PhD · 2025
(-)-Δ9-tetrahydrocannabinol and pregnancy: transporter-mediated tissue distribution and drug interactions - David Cohen · Nottingham · PhD · 2025
Magnetic resonance strategies for tracking immune cells - Khalid Ibrahim · EPFL · PhD · 2025
Intelligent and Self-Driving Microscopy of Protein Aggregation in Neurodegenerative Diseases
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