Library · Biology & lab methods
What Co-Immunoprecipitation Assays got wrong, from 23 theses
Summary written by a model from the records below: The records describe methodological limitations encountered when performing co-immunoprecipitation and related immunoprecipitation assays across various biological systems. Investigators frequently encountered difficulties involving the loss of transient complexes, buffer-induced disruption of physical interactions, antibody non-specificity, and insufficient target recovery for downstream analyses. 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 7 problems the authors left open, listed at the end.
Failure modes, by theses reporting them
Select a mode to jump to its records.
Weak and transient interactions dissociate during lysis and washing steps
Assays frequently failed or were abandoned because low-affinity, transient, or dynamic protein complexes dissociated during standard cell lysis and washing. Researchers repeatedly noted that gentle washing and non-denaturing conditions could not preserve these labile associations, prompting shifts toward proximity labeling techniques.
Tried and failed
co-immunoprecipitation of endogenous protein complexes applied to endogenous yeast protein-protein interactions. Outcome: no signal. Reason: endogenous interaction was too weak, transient, or disrupted during cell lysis despite recombinant binding
Parsing the Dual Roles of Cu/Zn Superoxide Dismutase (Sod1) in Oxidative Stress Protection and Redox Signaling · Georgia Tech · PhD
Considered and rejected
Considered and rejected: Rejected classical co-immunoprecipitation for identifying αVβ5 parasite partners due to low binding avidity and low parasite protein abundance.
A CRISPR-Cas9 screen for hepatocyte receptors for malaria parasite invasion · Oxford · Master's
Considered and rejected
Considered and rejected: Co-immunoprecipitation under non-denaturing conditions (NP40) was rejected/abandoned for identifying CD52 cis-ligands because weak/transient low-affinity glycan interactions could not be pulled down.
Adaptive immune dysfunction in acute decompensated cirrhosis · Imperial · PhD
Considered and rejected
Considered and rejected: Rejected standard immunoprecipitation (IP) in favor of BioID proximity labeling because IP requires gentle washing that allows non-specific contamination and loses transient interactions.
Investigating the Role of UL16 in the Nuclear Egress of Herpesvirus Capsids · Queen's University · Master's
Considered and rejected
Considered and rejected: Rejected co-immunoprecipitation due to transient phosphorylated interactions, switching to miniTurbo proximity labeling.
Regulation of dynamic front-rear cell polarity by the Frz chemosensory system in Myxococcus xanthus · University of Marburg · PhD
Considered and rejected
Considered and rejected: Rejected standard immunoprecipitation alone in favor of proximity-dependent biotinylation (BioID) to identify transient or weak interactors of the VIGSSK domain.
Cellular and biochemical characterisation of ERCC6L2 · Oxford · PhD
Considered and rejected
Considered and rejected: Rejected co-immunoprecipitation for validating ETCC-PAF interactions due to inconsistent results, cross-BET dimerization artifacts via motif B, and low sensitivity for transient complexes; recommended baculovirus/insect cell purification instead.
Structure-Function Analysis Of Bet Proteins In Transcription · Penn · PhD
Considered and rejected
Considered and rejected: Standard co-immunoprecipitation was deemed insufficient on its own due to unspecific binding after cell lysis and failure to capture weak/transient transport interactions, prompting the adoption of in situ proximity labeling and sptPALM.
The path and regulation of the type III secretion effector export · University of Marburg · PhD
Considered and rejected
Considered and rejected: Rejected yeast two-hybrid, bacterial two-hybrid, co-immunoprecipitation, and FRET for mapping anaerobic Fd interactions due to oxygen sensitivity, high salt wash interruption of transient complexes, and lack of oxygen-independent fluorophores.
Insights into the electron transport proteins essential for nitrogen fixation · University of Marburg · PhD
Buffer components and harsh extraction conditions disrupt complex integrity or stability
Chemical additives such as detergents and reducing agents in lysis buffers directly disrupted protein interactions that depended on hydrophobic associations or disulfide bonds. In other instances, protein denaturation compromised necessary antibody binding or the absence of proteasome inhibitors allowed target degradation.
Tried and failed
in vitro co-immunoprecipitation applied to hydrophobic membrane protein interaction. Outcome: no signal. Reason: protein hydrophobicity caused aggregation leading to marginal elution
Self-recognition signaling and ecological variation in the bacterial pathogen Proteus mirabilis · Harvard · PhD
Tried and failed
immunoprecipitation of lipid-modified protein applied to membrane-associated kinase complex identification. Outcome: no signal. Reason: failed to capture stable membrane compartment associations or strong direct interactor binding
Tried and failed
co-immunoprecipitation without proteasome inhibition applied to detecting transient protein-protein interactions. Outcome: no signal. Reason: Target protein underwent rapid proteasomal degradation in the absence of a proteasome inhibitor.
MFSD7C: A Solute Carrier Linking Heme and Calcium in Mitochondrial Energy Metabolism · MIT · PhD
Tried and failed
detergent co-immunoprecipitation applied to detecting soluble protein family hetero-oligomerisation. Outcome: no signal. Reason: detergent presence disrupted weak soluble interactions or the proteins do not hetero-oligomerise in solution
The Role of Membrane Binding of α-Synuclein in the Central and Enteric Nervous System · Cornell · PhD
Tried and failed
co-immunoprecipitation with reducing agent in lysis buffer applied to protein-protein interaction detection. Outcome: no signal. Reason: reducing agent in lysis buffer disrupted the protein-protein interaction complex
Modulation of innate immune signalling by salmonella effector SteE · Imperial · PhD
Considered and rejected
Considered and rejected: Rejected 2C (complex capture) because protein denaturation can compromise antibody binding needed for downstream immunoprecipitation.
Investigating the role of MYCN in alternate splicing in MYCN-amplified neuroblastoma · University College Dublin · PhD
Antibody cross-reactivity and non-specific binding cause severe background contamination
Assays were impaired by antibodies displaying non-specific binding, carryover, or poor affinity for specific target epitopes. This produced substantial background signals in control lysates, yielded abundant contaminant proteins such as keratins, or failed to enrich the intended targets.
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
immunoprecipitation-mass spectrometry and multimer structure prediction applied to detecting plant protein-protein interactions. Outcome: no signal. Reason: IP-MS yielded only keratin contaminants and structure prediction crashed on full-length protein complexes
The role of h3k27me3 reprogramming in arabidopsis development · Imperial · PhD
Tried and failed
chromatin immunoprecipitation with endogenous target antibodies applied to endogenous GLIS2 transcription factor. Outcome: no signal. Reason: antibodies failed to specifically enrich the endogenous transcription factor protein
Transcriptional regulators in stem cell biology · 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 RL2 antibody for direct immunoprecipitation/enrichment because it requires specific tertiary epitopes and exhibits poor affinity for proteins under 50 kDa.
Untersuchungen zur O-GlcNAc Modifikation von Plasmodium falciparum dem Erreger der Malaria tropica Investigation of the O-GlcNAc modification in the malaria parasite Plasmodum falciparum · University of Marburg · PhD
Chemical crosslinking and sample preparation reduce recovery yields and mask epitopes
Formaldehyde and glutaraldehyde crosslinking combined with sonication caused epitope occlusion and severe sample loss. These protocols resulted in recovery yields that were insufficient for downstream mass spectrometry or sequencing applications.
Considered and rejected
Considered and rejected: Rejected glutaraldehyde-based co-immunoprecipitation MS (IP-MS) in favor of whole-membrane extraction with MS-cleavable crosslinker DSSO due to protein loss and detection failures.
Investigating proteins that influence membrane-associated germination processes in Bacillus subtilis spores · Virginia Tech · Doctorate
Tried and failed
chromatin immunoprecipitation combined with mass spectrometry applied to chromatin-bound protein complexes. Outcome: data insufficient. Reason: insufficient protein yield harvested without the ability to amplify unamplifiable protein targets
PROTEIN-PROTEIN INTERACTION NETWORK PERTURBATIONS CAUSED BY ONCOGENIC FUSION EVENTS · Cornell · PhD
Tried and failed
chromatin immunoprecipitation with formaldehyde crosslinking and sonication applied to large nuclear pore complex proteins. Outcome: no signal. Reason: antibody epitope occlusion caused by chemical crosslinking and sonication-induced shearing
Nuclear Pore Component Mtor Regulates Levels Of Dosage Compensated Gene Expression In Drosophila · Penn · PhD
Tried and failed
Chromatin immunoprecipitation sequencing using tagged protein applied to overexpressed fluorescently-tagged transcription factor. Outcome: data insufficient. Reason: Insufficient immunoprecipitated DNA yield for sequencing despite extended cross-linking and increased input tissue
The role of h3k27me3 reprogramming in arabidopsis development · Imperial · PhD
Left open by the authors
Problems the authors named and did not get to.
Left open
Perform co-immunoprecipitation assays of Lrp13b with clathrin, adaptor protein complex, or disabled to determine involvement in clathrin-mediated endocytosis. Blocker: Requires a wet biology laboratory, biochemical reagents, and physical tissue/cell samples for co-immunoprecipitation assays.
A novel low-density lipoprotein receptor-related protein protects against environmental teratogens · UT Austin · PhD
Left open
Investigate specific protein-protein interactions between TerC proteins (MeeF, MeeY) and secretosome components (SecDF, PrsA, FtsH). Blocker: Requires a wet lab and molecular biology assays (e.g., co-immunoprecipitation, bacterial two-hybrid) to test protein interactions.
THE FUNCTION OF TERC PROTEINS IN METAL HOMEOSTASIS · Cornell · PhD
Left open
Test whether NiV F or G interacts directly with mitochondrial fission proteins like Drp-1 via co-immunoprecipitation or two-hybrid screening. Blocker: Requires a wet lab, cell cultures, and biological assays for co-immunoprecipitation or yeast two-hybrid screening.
VIRUS-HOST INTERACTIONS AND THE IMPACT ON HOST CELLULAR MECHANISMS · Cornell · PhD
Left open
Identify the in vivo protein interaction partner of ClbP in microcin production using alternative growth media or co-immunoprecipitation. Blocker: Requires wet lab facilities, biological cell cultures, and co-immunoprecipitation assays
Exploring the mechanisms and structures of N-acyl-D-Asn prodrug peptidases · Harvard · PhD
Left open
Identify physical binding partners of Arabidopsis ECT8 by performing co-immunoprecipitation followed by mass spectrometry. Blocker: Requires wet-lab facilities, plant samples, and mass spectrometry apparatus to perform co-IP-MS.
THE EPITRANSCRIPTOMIC REGULATION OF STRESS: ROLE OF N6-METHYLADENOSINE IN ABIOTIC STRESS RESPONSE · Penn · PhD
Left open
Determine if STING colocalizes and interacts with TLR8 and IKKε in the Myddosome using immunofluorescence microscopy and co-immunoprecipitation. Blocker: Requires a wet laboratory, physical cell cultures, and biological assays (immunofluorescence microscopy and co-immunoprecipitation).
ELUCIDATING THE INTRICATE CROSSTALK BETWEEN PATTERN RECOGNITION RECEPTORS AND HOST-MICROBIOME INTERACTIONS FOR DISEASE THERAPEUTICS · Cornell · PhD
Left open
Screen for interacting protein or metabolite partners of SCD1 using proximity-based co-immunoprecipitation. Blocker: Requires wet lab facilities, biological reagents, and proximity-based co-immunoprecipitation experimental assays.
The Role of Ring Finger Protein 213 in Fatty Acid-induced Lipotoxicity · Harvard · PhD
Who has worked on this
Authors of the public theses cited on this page, most-cited first.
- Charlie Keyzor · Imperial · PhD · 2023
The role of h3k27me3 reprogramming in arabidopsis development - Gerardo Estrada Zavala · Harvard · PhD · 2026
Exploring the mechanisms and structures of N-acyl-D-Asn prodrug peptidases - Katherine Pintor · University of Marburg · PhD · 2025
The path and regulation of the type III secretion effector export - Holly Gabriela Addison · University of Marburg · PhD · 2025
Insights into the electron transport proteins essential for nitrogen fixation - Karim Omar · Cornell · PhD · 2024
PROTEIN-PROTEIN INTERACTION NETWORK PERTURBATIONS CAUSED BY ONCOGENIC FUSION EVENTS - Bixi He · Cornell · PhD · 2024
THE FUNCTION OF TERC PROTEINS IN METAL HOMEOSTASIS - Tong Liu · Imperial · PhD · 2024
Adaptive immune dysfunction in acute decompensated cirrhosis - Hembly Rivas · Harvard · PhD · 2024
B-MYB and FOXM1 drive cell division through cooperative regulation of cell cycle genes. - Franziska Müller · University of Marburg · PhD · 2024
Regulation of dynamic front-rear cell polarity by the Frz chemosensory system in Myxococcus xanthus - Laura Maugeri · University College Dublin · PhD · 2024
Investigating the role of MYCN in alternate splicing in MYCN-amplified neuroblastoma - Haewon May Byun · Cornell · PhD · 2023
VIRUS-HOST INTERACTIONS AND THE IMPACT ON HOST CELLULAR MECHANISMS - Karla Garcia-Martinez · Cornell · PhD · 2023
ELUCIDATING THE INTRICATE CROSSTALK BETWEEN PATTERN RECOGNITION RECEPTORS AND HOST-MICROBIOME INTERACTIONS FOR DISEASE THERAPEUTICS
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