Chapter Four · failure evidence
What Chromatography & Protein Purification got wrong, from 77 dissertations
The records document numerous experimental failures and operational trade-offs encountered during protein purification and chromatographic separation protocols. These difficulties frequently stem from non-specific matrix interactions, target aggregation or instability, insufficient resolving power between similar species, and impaired affinity tag capture. These records come from PhD theses at 21 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.
Affinity tag inaccessibility, weak resin binding, or elution failure prevents target recovery
Target proteins and assembled complexes frequently fail to bind affinity columns when tags are sterically buried, mutated, or possess insufficient binding affinity for the complementary matrix. In other instances, immobilized targets cannot be released without denaturants, suffer excessive wash loss, or show adverse interactions between elution reagents and catalytic metal centers.
Tried and failed
recombinant bacterial expression with affinity chromatography applied to bacterial outer membrane proteins. Reason: protein aggregated into insoluble inclusion bodies and failed affinity column binding
The interaction of glycosylated major outer membrane protein of Campylobacter jejuni with natural and synthetic ligands · University of Nottingham Repository
Lost to a baseline
Affinity chromatography using a CPV-coupled Sepharose column resulted in high sample loss and lower yields compared to mixed-mode ceramic hydroxyapatite purification
Tried and failed
immobilized metal affinity chromatography with cobalt resin applied to recombinant his-tagged membrane protein purification. Reason: weak His-tag binding caused almost total protein loss in flow-through and early wash steps
Tried and failed
immobilized metal affinity chromatography of intact complexes applied to membrane protein complexes with terminal tags. Reason: engineered affinity tags were buried within the assembled complex and inaccessible to the resin
Directionality of electron transfer within Photosystem I complex · Iowa State
Tried and failed
immobilized metal affinity chromatography for purification applied to secreted recombinant proteins in fermentation broth. Outcome: no signal. Reason: failed to detect or bind the tagged protein from the culture broth
Tried and failed
subtractive IMAC purification after affinity tag cleavage applied to recombinant protein purification. Reason: cleaved target protein retained unexpected intrinsic affinity for immobilized metal affinity chromatography resin
Tried and failed
affinity purification using C-terminal affinity tag applied to multisubunit ribonucleoprotein complex lacking accessory subunit. Reason: the subunit tag interfered with assembly or complex was unstable without the missing subunit
Structures of Class 1 CRISPR-Cas surveillance complexes · UT Austin
Tried and failed
immobilized cobalt affinity chromatography purification applied to polyhistidine-tagged recombinant toxin protein. Reason: Weak target protein binding caused substantial loss in column flow-through and low imidazole wash fractions
Characterising the protein-protein interactions of TacTA1-3 toxin-antitoxin family · Imperial
Considered and rejected
Considered and rejected: Purification tags MBP and His6 for recombinant Mymx ectodomain due to failure to release protein without detergents
Myogenic Effectors and Disease · DSpace at UTSWMED
Considered and rejected
Considered and rejected: Rejected standard His-tag/Ni-NTA purification with imidazole elution due to interference with the catalytic non-heme iron center.
Tried and failed
iminobiotin affinity chromatography purification applied to monoavidin surface-engineered extracellular vesicles. Reason: engineered monoavidin had insufficient binding affinity to iminobiotin resin at binding pH, leading to negligible recovery
Modular assembly of bacterial outer membrane vesicle-based vaccines and therapeutics · Cornell
Considered and rejected
Considered and rejected: Rejected transferring standard affinity resin operating parameters directly to membrane chromatography without re-optimizing buffer ionic strength and pH due to severe unbinding/recovery failure.
Evaluating Improvements and Challenges in Affinity Chromatography for AAV Purification · JScholarship
Lost to a baseline
Protein G purification resulted in significantly lower intact protein yield (62.5% loss for KbIg, 67.0% for A2Ig) compared to standard Sepharose-NP/NIP extraction (26% loss for KbIg, 44.9% for A2Ig)
OPTIMIZATION OF METHODS FOR MHC-IG SYNTHESIS, PURIFICATION, AND EVALUATION FOR T CELL IMMUNOENGINEERING · JScholarship
Considered and rejected
Considered and rejected: Discarded Strep-tag affinity chromatography for integrin purification because it diluted protein without increasing purity
Structural Studies of N-Acetylmuramic Acid Kinases from the Bacterium Tannerella forsythia and of the Human Adenovirus Penton Base-Integrin Entry Receptor Complex · Publikationssystem UB Tuebingen
Inadequate resolution, co-elution, or severe sample loss occurs during ion exchange chromatography
Ion exchange methods fail to separate target proteins from cleavage tags, conjugates, or impurities when surface charge differentials are insufficient. Furthermore, altered surface charges cause premature flow-through loss, while extreme retention or low salt conditions lead to irreversible matrix binding and heavy protein precipitation.
Tried and failed
inclusion body prep with ion exchange chromatography applied to multiply modified recombinant proteins. Reason: standard chromatography steps lacked sufficient resolving power to yield pure modified target protein
Tried and failed
anion exchange chromatography applied to protein-small molecule conjugate separation. Reason: insufficient charge differential caused co-elution of unmodified and modified protein
Modification of Biopolymers Using Palladium Oxidative Addition Complexes · MIT
Tried and failed
mixed-mode ion-exchange reverse-phase chromatography applied to hydrolyzed sulfonated azo dyes. Reason: irreversible adsorption to stationary phase prevented analyte elution despite strong gradient
Monitoring for sulfonated azo dyes and their degradation products in industrial and municipal wastewater · Virginia Tech
Tried and failed
anion exchange chromatography with salt gradient applied to cleaved fusion protein separation. Reason: target protein and cleavage tag co-eluted due to similar charge characteristics
C2 domains of dysferlin and synaptotagmin-1: Structure and mechanics · Texas Tech
Tried and failed
single affinity pulldown and ion exchange chromatography applied to multi-subunit protein complex purification. Reason: failed to yield stoichiometric complex, causing non-stoichiometric subunit accumulation
A single-molecule approach to understanding sister chromatid cohesion establishment · Imperial
Tried and failed
anion exchange chromatography using standard binding conditions applied to truncated protein purification. Reason: truncation reduced net negative charge, preventing binding at standard ionic strength and causing flow-through loss
Purification and Functional Analysis of XRCC4’s C-Terminal Tail in DNA Binding Efficiency · UT Austin
Tried and failed
ion-exchange chromatography for recombinant protein purification applied to recombinant bacterial beta-lactamase enzyme. Reason: produced heavily contaminated samples with minimal target protein yield
Tried and failed
elution using specific chaotropic displacing counterions applied to anion exchange chromatography of nucleic acids. Reason: Counterions caused either premature dead-volume elution or extreme retention and co-elution
Improving bioanalytical methods for nucleic acids with chemically tuned separations · Iowa State
Considered and rejected
Considered and rejected: Omitted ion exchange chromatography after IMAC as it failed to enrich singlet pentamers
Structural Studies of N-Acetylmuramic Acid Kinases from the Bacterium Tannerella forsythia and of the Human Adenovirus Penton Base-Integrin Entry Receptor Complex · Publikationssystem UB Tuebingen
Considered and rejected
Considered and rejected: Anion exchange chromatography (AEX) was omitted for His6-TEV-GGG-16E7 purification due to excessive protein loss.
Cellular and Intraviral Interaction Partners of Papillomavirus E6 Protein · Publikationssystem UB Tuebingen
Considered and rejected
Considered and rejected: Ion exchange chromatography (IEC) and Hydroxyapatite chromatography (HAC) due to high salt concentrations corroding pumps/columns and inability to resolve all six proteins in one run
Quantification of major bovine milk proteins · Iowa State
Considered and rejected
Considered and rejected: Rejected ion exchange chromatography in low salt buffers for hMiro1 and hMiro1(R450C) due to heavy protein precipitation
Structural Analysis of Protein Complexes · Publikationssystem UB Tuebingen
Considered and rejected
Considered and rejected: Rejected DEAE-C anion exchange chromatography for peptide purification due to substantial loss of peptide recovery, replacing it with cold 1:1 Et2O:hexane precipitation before HPLC.
Biosynthetic and Chemical Investigation of Lipid II-Binding Antimicrobials. · DukeSpace
Non-specific binding and host protein contamination compromise affinity chromatography purity
Affinity matrices like immobilized metal affinity resins frequently capture sticky host cell proteins alongside the desired recombinant target. This persistent non-specific interaction leads to contaminated elution fractions, co-purification of unwanted cellular impurities, and degraded target purity.
Tried and failed
affinity purification without heat-denaturation contaminant precipitation applied to sticky recombinant bacterial effector protein. Reason: protein stickiness caused co-purification of host contaminants and multiple gel filtration peaks
Structural and Functional characterisation of SteE, a Salmonella type III secretion system effector · Imperial
Tried and failed
immobilized metal affinity chromatography applied to recombinant bacterial protein purification. Reason: co-elution of an unexpected high molecular weight host protein impurity with the target
Cross-Species Analysis of YTH Domain Proteins from H. sapiens and A. ricinus · UT Austin
Considered and rejected
Considered and rejected: Rejected Ni-NTA resin in favor of TALON (Co2+) affinity chromatography due to poor purity and non-specific binding
Investigating tRNA Release from the Bacterial Ribosome · Penn
Tried and failed
His-tag affinity purification from membrane fractions applied to heterologously expressed bacterial membrane transporters. Outcome: no signal. Reason: Membrane extractions yielded excessive non-specific binding and failed to isolate the full-length target proteins.
Tried and failed
immobilized metal affinity chromatography using imidazole gradient applied to recombinant histidine-tagged protein purification. Reason: leaky uninduced expression and persistent non-specific co-elution across imidazole concentrations
Tried and failed
polyhistidine tag affinity purification and protease cleavage applied to membrane protein purification from mammalian cells. Reason: dominant host protein contamination and incomplete 3C protease cleavage within 24-48 hours
Structure of a silk moth fructose receptor: an ion channel involved in insect chemosensation · Harvard
Tried and failed
C-terminal epitope tagging for affinity purification applied to protein complex co-enrichment. Outcome: no signal. Reason: Enriched non-specific ribonuclear proteins instead of intended complex members
Investigation of E3 ligase ligands for targeted protein degradation · Harvard
Tried and failed
affinity chromatography and size exclusion chromatography applied to fluorophore labeled membrane protein purification. Reason: host membrane proteins containing reactive cysteines co-purified and labeled alongside the target protein
Exploring the Structural Dynamics of Bacterial Chemotaxis · MIT
Lost to a baseline
HisTrap HP purification produced higher gross yield (3 mg) but was contaminated compared to the lower yielding (100–282 µg) Strep-Tactin XT purification.
Structural and functional characterisation of the human vitamin c transporters · Imperial
Considered and rejected
Considered and rejected: Purification utilizing 6x His tags (both N- and C-terminal) on Ni/NTA resin due to excessive non-specific background binding.
Residual Neuropathy target esterase activity defines the PNPLA6 disorder spectrum · JScholarship
Considered and rejected
Considered and rejected: Initial standard His-tag nickel resin purification protocol (with 1-hour incubation and standard wash volume) yielded significant impurities and was revised
Rationally-Engineered Protein Therapeutics for the Treatment of Ewing Sarcoma · Harvard
Considered and rejected
Considered and rejected: Rejected Ni-NTA affinity chromatography for large-scale SMALP purification because target purity decreased with increasing scale, choosing Twin-Strep/Strep-Tactin instead
Biochemical Characterization of Glycan Assembly Pathway Enzymes · MIT
Protein unfolding, aggregation, or complex dissociation occurs during size exclusion chromatography
Labile protein complexes and destabilized constructs often dissociate into individual subunits or unfold when subjected to size exclusion chromatography. Inadequate cosolvents, varying ionic environments, and prolonged separation runs frequently induce misfolding and drive target proteins into void volume aggregates.
Tried and failed
recombinant full-length protein expression and purification applied to bacterial effector protein SteC. Outcome: unstable. Reason: severe protein aggregation causing elution in size exclusion chromatography void volume
Studying the mechanism of Salmonella effector protein, SteC · Imperial
Tried and failed
size-exclusion chromatography mass spectrometry for complex isolation applied to weakly binding monomeric peptide-protein complexes. Outcome: no signal. Reason: weak binding affinity caused complex dissociation during column separation
Affinity Maturation of Peptides to Bind Protein-Protein Interfaces · MIT
Tried and failed
size exclusion chromatography in low-cosolvent buffer applied to destabilized recombinant kinase domain variants. Outcome: unstable. Reason: insufficient glycerol concentration failed to prevent protein aggregation and void volume elution
Biochemical characterization of EGFR exon 20 insertion variants and their inhibitor sensitivities · Harvard
Tried and failed
recombinant protein co-expression for complex purification applied to heteromeric protein complex assembly. Outcome: unstable. Reason: complex dissociated during size-exclusion chromatography
Structural and biochemical studies of RAD51 filament modulators · Imperial
Tried and failed
co-purification via affinity and size-exclusion chromatography applied to multimeric ribonucleoprotein-associated effector complexes. Reason: effector subunit lacked sufficient binding affinity to remain stably associated during chromatography steps
Structures of Class 1 CRISPR-Cas surveillance complexes · UT Austin
Considered and rejected
Considered and rejected: Rejected inverse-size exclusion chromatography (ISEC) using spherical proteins (ovalbumin, haemoglobin) because misfolding/aggregation under varying mobile phase compositions caused unreliable results.
Fundamentals of peptide adsorption in reversed-phase liquid chromatography · Imperial
Considered and rejected
Considered and rejected: Abandoned co-expression and size-exclusion chromatography strategies for FIGNL1-RAD51 complex purification due to resin interaction and complex dissociation
Structural and biochemical studies of RAD51 filament modulators · Imperial
Tried and failed
size-exclusion chromatography under varying ion concentrations applied to measuring ion-dependent protein trimerization threshold. Outcome: unstable. Reason: protein instability and monomer unfolding occurred at low ion concentrations
Structural and biochemical investigations of collagen-I trimerization · MIT
Considered and rejected
Considered and rejected: Rejected size exclusion chromatography at room temperature for rDps purification due to protein cleavage/instability, switching to gradient pH elution on Ni-NTA resin.
Identification and characterisation of of vaccine antigens for Klebsiella pneumoniae · Research Repository UCD
Tried and failed
multi-step chromatographic protein purification applied to recombinant kinase cascade enzymes. Reason: extended purification time caused protein misfolding and loss of catalytic activity
Non-specific stationary phase adsorption causes severe sample loss in size exclusion chromatography
Target proteins, multimeric complexes, and nanomaterials frequently exhibit strong hydrophobic or electrostatic interactions with size exclusion resin matrices and filtration media. These non-specific interactions lead to irreversible column sticking, substantial carryover, and drastic or complete loss of recovered sample.
Tried and failed
size exclusion chromatography purification applied to recombinant multiprotein complex. Reason: severe sample loss caused by non-specific matrix sticking and aggregation
Tried and failed
size exclusion chromatography applied to purified multi-protein complex. Reason: hydrophobic interactions with the stationary phase matrix caused irreversible protein adsorption and sample loss
Characterization of the DPP8/9 Pyroptotic Checkpoints · Cornell
Tried and failed
size exclusion chromatography after ion exchange applied to multimeric protein complex purification. Reason: hydrophobic interactions with the column matrix caused complete sample loss
Molecular mechanisms for the regulation and assembly of the NLRP1 and CARD8 inflammasomes · Harvard
Tried and failed
membrane filtration and size exclusion chromatography applied to nanoparticle purification. Reason: severe particle agglomeration and material loss caused by filter adherence
Integrated Diamond-Based Devices For Quantum Sensing And Communication · Penn
Tried and failed
size exclusion chromatography with surfactant mobile phase applied to nanoparticle and organic matter mixtures. Reason: severe irreversible stationary phase adsorption and sample carryover
Investigation of zinc nanoparticles in soil environments · University of Nottingham Repository
Tried and failed
size exclusion chromatography with standard organic columns applied to deprotected cationic polyesters. Reason: strong irreversible interactions between protonated amine groups and the polystyrene-divinylbenzene stationary phase
Multifunctional polyester-based stimuli-responsive polymeric nanoparticles for therapeutic delivery · Imperial
Considered and rejected
Considered and rejected: Rejected standard size-exclusion chromatography for NLRP1-DPP9 complex purification due to complete sample loss from hydrophobic column interactions, relying instead on Mono Q anion exchange
Characterization of the DPP8/9 Pyroptotic Checkpoints · Cornell
Considered and rejected
Considered and rejected: Rejected size exclusion chromatography (SEC) for large-scale purification of CD630_08730 because it caused unacceptable loss of target protein yield.
Development of an oral vaccine against Clostridioides difficile · University of Nottingham Repository
Considered and rejected
Considered and rejected: Rejected size exclusion chromatography (gel filtration) after affinity purification because recovery yield dropped to ~10% without fully removing degradation products.
Study of glyceraldehyde-3-phosphate oxidoreductases from archaea and bacteria · University of Nottingham Repository
Overlapping hydrodynamic sizes and low throughput limit size exclusion chromatography separation
Size exclusion columns frequently fail to separate target molecules from oligomers, degradation products, or liposomal conjugates with similar hydrodynamic dimensions. In addition, these separation protocols suffer from slow flow rates, restricted sample loading capacities, and extended run times that hinder throughput.
Tried and failed
size exclusion and density gradient centrifugation applied to protein purification of degradation products. Reason: failed to resolve impurities and degradation products with similar size or hydrodynamic properties
Tried and failed
size-exclusion chromatography multimer fractionation applied to heterogeneous multi-domain protein complexes. Reason: columns failed to separate oligomeric species across different resins and construct variants
Structural and Biophysical Characterization of the Myocilin-OLF Domain Under Various Stressors · Georgia Tech
Tried and failed
size exclusion chromatography purification applied to micellar amphiphilic conjugates from liposomes. Reason: insufficient size difference to resolve free or micellar amphiphilic conjugate from PEGylated liposomes
Analysis of the properties of phospholipid bilayers for drug delivery using solid-state NMR · Imperial
Lost to a baseline
qEV size-exclusion chromatography resulted in lower RNA recovery and higher Ct values compared to ultracentrifugation, PEG precipitation (ExoQuick), and membrane affinity (exoEasy).
Evaluation of Cell-Free RNA as Minimally Invasive Biomarkers of Target Engagement · Harvard
Lost to a baseline
Size-exclusion chromatography (SEC) on Sephadex LH-20 with aqueous methanol required a 2- to 3-fold increase in run time (24 h vs 30-60 min flash chromatography) and suffered from low loading capacity (few hundred mg) and loss of stationary phase during packing cycles.
Microcystins as Lead Structures for Anticancer Agents · Publikationssystem UB Tuebingen
Tried and failed
size exclusion chromatography purification applied to chemical synthesis product. Outcome: too slow. Reason: resulted in significant decreases in synthesis throughput
Toward a Sensor for DNA Supercoiling · Harvard
Considered and rejected
Considered and rejected: Rejected protein A affinity chromatography and size-exclusion chromatography for separating WT Fab from Fc fragments post-papain digestion due to co-elution, switching to anion exchange chromatography at pH 8.
Site-specific conjugation for the synthesis of radiopharmaceuticals · Imperial
Severe sample insolubility and uncontrolled aggregation prevent liquid chromatographic purification
Hydrophobic peptides, rigid synthetic polymers, and intrinsically disordered proteins frequently aggregate or precipitate out of solution under non-denaturing conditions. This poor solubility prevents stable column loading, obstructs dissolution in compatible mobile phases, and causes extensive protein smearing or precipitation.
Tried and failed
size exclusion chromatography molecular weight characterisation applied to poorly soluble rigid polymers. Reason: polymer was only soluble in solvents incompatible with standard chromatographic equipment
Water vapor and CO₂ sorption and transport in carbon molecular sieve membranes and moisture swing materials · UT Austin
Tried and failed
anti-solvent precipitation for polymer purification applied to functionalised polyelectrolyte polymers. Reason: precipitation in organic solvent significantly reduced polymer solubility, preventing downstream layer-by-layer assembly
Tried and failed
native protein purification with affinity tags applied to intrinsically disordered protein purification. Outcome: unstable. Reason: extensive protein degradation and aggregation occurred under non-denaturing conditions
THE NEUROSPORA CRASSA CIRCADIAN CLOCK: AN ORDERLY LOOP DRIVEN BY A DISORDERED CONDUCTOR · Cornell
Tried and failed
his-tag affinity purification without heat denaturation applied to self-assembling fusion proteins. Reason: uncontrolled oligomerization caused protein smearing during electrophoretic separation
Tried and failed
solid-phase peptide synthesis and chromatographic purification applied to hydrophobic dipeptide repeat proteins. Reason: extreme insolubility and aggregation prevented purification despite successful chemical synthesis
The Discovery and Chemical Synthesis of Peptides and Proteins that Cross Biological Barriers · MIT
Tried and failed
solution-phase purification and characterization applied to highly hydrophobic oligopeptoids. Reason: extreme hydrophobicity prevented dissolution required for liquid-phase purification and characterization
PRECISION ENGINEERING OF SMALL MOLECULES AND OLIGOMERS FOR ADVANCED TECHNOLOGIES · Cornell
Considered and rejected
Considered and rejected: Rejected purification Method A due to persistent ~10% contamination and protein precipitation at pH 4.5 within 12 hours.
Characterization of specific aptamer interactions with NGAL · Iowa State University Digital Repository
Left open by the authors
Problems the authors named and did not get to.
Left open
Demonstrate direct metal transfer or stable complex formation between ZagA and FolE using size exclusion chromatography in the presence of ZTP. Blocker: Requires wet lab biochemistry facilities, purified proteins (ZagA, FolE), ZTP, and size exclusion chromatography instrumentation.
METAL TRAFFICKING DURING LIMITATION AND EXCESS IN BACILLUS SUBTILIS · Cornell
Left open
Optimize sample preparation using size-exclusion chromatography and collect cryo-EM particle data to reconstruct the DNA mismatch repair initiation complex in RELION. Blocker: Requires wet lab facilities for protein purification, chromatography, and access to a cryo-electron microscope
The Crosstalk Between DNA Mismatch Repair and Replication · DSpace at UTSWMED
Left open
Evaluate enhanced coagulation on isolated low and high molecular weight wastewater fractions to determine size-specific removal mechanisms. Blocker: Requires a wet chemistry laboratory, wastewater samples, fractionation membranes, and coagulation experimental apparatus.
Enhanced Coagulation Process for Dissolved Organic Nitrogen (DON) Removal from Wastewater · unevada
Left open
Characterize intermediate fractions between cell debris and extracellular vesicle sizes, and define extracellular vesicle compositions from MSC lysates. Blocker: Requires wet lab equipment, cell cultures, and biological fractionation/characterization assays.
Left open
Implement cation exchange chromatography targeting N- and C-terminal globular domains for recombinant lubricin purification to prevent loss during high-salt wash. Blocker: Requires a wet lab and physical chromatography equipment/protein samples
Left open
Develop an anion ion chromatography method to separate and quantify polythionates for tracking sulfite reservation in amine scrubbing campaigns. Blocker: Requires a physical analytical chemistry wet lab and an ion chromatography instrument with chemical standards.
Understanding and mitigating amine oxidation caused by NO₂ in scrubbing to capture CO₂ · UT Austin
Left open
Develop an extraction and secondary chromatographic method using polar sorbents or anion exchange resins to retain short-chain PFAS analytes. Blocker: Requires an analytical chemistry wet lab, chromatography equipment, and chemical standards.
Evaluating "Forever Chemicals" from Soup to Nuts: Sample Preparation, Targeted Analysis, and Suspect Screening for the Characterization of Per- and Polyfluoroalkyl Substances (PFAS · DSpace at SUNY Buffalo
Left open
Optimize glycopeptide enrichment, purification, and ionization methods for mass spectrometry-based glycoproteomics analysis. Blocker: Requires wet lab access, chemical reagents, and physical mass spectrometry instruments for sample preparation and ionization
Left open
Test shaking instead of sonication during extraction of PFAS from aluminum foil cascade impactor filters to prevent foil disintegration. Blocker: Requires a wet chemistry analytical lab, chemical reagents, and chromatography/mass spectrometry equipment (GC-MS, UPLC-MS)
Per and Polyfluoroalkyl Substances (PFAS) in Indoor Airborne Particulate Matter · Carleton University Institutional Repository
Left open
Synthesize and test phosphonium, pyridinium, and ammonium ionic liquids with non-Tf2N anions using inverse gas chromatography to measure separation selectivity. Blocker: Requires a wet chemistry lab, chemical synthesis of ionic liquids, and gas chromatography apparatus.
Inverse gas chromatography for crucial separations using ionic liquids · UT Austin
Checking a claim in this area?
We can run the same search on any method or claim. If nothing turns up, we will say so, and that proves nothing on its own.