Research method
Western Blot
12 studies
A western blot separates proteins by electrophoresis, transfers them to a membrane, and detects a target with antibodies (or a tagged fusion). The output is a band whose size, abundance, or mobility shift reports presence, cleavage, ubiquitination, or glycosylation of that protein. A blot is not a subcellular map unless the sample was first fractionated, and antibody specificity is part of the result.
Molecular biologists use westerns when they need to know whether a protein is there, modified, or physically associated after a pull-down. It answers 'did this species change size or amount under this perturbation?' Its main limitation is that a band does not prove localisation or in-vivo dynamics, and some papers in this list use blots only as a supporting readout beside genetics, proximity labelling, or viral titres.
Studies
12
Findings
5
7 supporting · 0 challenging · 0 qualifying citations
Open tensions
2
Currently
What we know
- Caveolar-coat purification used quantitative composition to show caveolins and cavins in one complex that excludes EHD2/pacsin 2, with Cavin 1 a core component and evidence for trimers. Western or blot-style detection of coat subunits supports stoichiometry; it is not a live-cell movie of every tissue's caveolae.
- Tagged tobacco N and TMV p50 co-expressed in N. benthamiana associated in the cytoplasm; the TIR domain of N was critical for that association, and co-expression triggered hypersensitive-response cell death in two days. Domain mapping of a physical association is a classic immunoblot/co-IP use, not an atomic structure of the complex.
- USP8 limits Smoothened ubiquitination: RNAi and biochemical readouts showed less ubiquitin on Smo, stronger Hedgehog signalling, and Hh-stimulated Smo–USP8 interaction. Ubiquitin blots here are a modification assay tied to trafficking, not a complete ligase identification.
- SARS-CoV-2 spike LYQD mutants changed S2 glycosylation mobility, and palmitoylation at cysteine clusters I/II was required for efficient particle production, Golgi/plasma-membrane targeting, and ACE2-mediated fusion (2-bromopalmitate inhibition). Mobility shifts on S2 are a western-style glycosylation readout; much of the work uses spike pseudoviruses rather than full-virus genetics for every assay.
- Not every tagged paper is a western-first study. APEX2 maps (22 OMM, 72 ERM proteins) are mass-spectrometry inventories; Wnt5a work uses purified ligand and receptor context to turn β-catenin–TCF signalling on or off; Cas13a plant antiviral work measures GFP and viral accumulation after targeting TuMV regions. Blots may appear as supporting assays, not as the experimental core.
Largest unresolved question
Westerns here answer different biochemical questions. Caveolar work asks which subunits co-purify; N–p50 asks which domain is required for association and HR; USP8 asks whether Smo is ubiquitinated; spike work asks whether glycan occupancy and palmitoylation change S2 mobility and trafficking. A darker band is not a universal 'more protein' story across those designs.
Procedure
How it works
The main stages of the method. Specific protocols and analysis choices vary between studies.
Separate proteins by electrophoresis
A lysate, pull-down, or fraction is run on a gel so species resolve by size (and sometimes charge). What is loaded is already a chosen extract; a whole-cell lysate will not report a compartment unless it was first fractionated.
Transfer to a membrane
Proteins are moved onto nitrocellulose or PVDF so they can be probed as a two-dimensional blot. Transfer efficiency is part of the result; a missing band can be a failed transfer rather than absence.
Probe with an antibody or a tag
An antibody against the target, or a tagged fusion, produces a band. Antibody specificity is the single largest determinant of whether that band is the intended protein; a cross-reactive antibody yields a clean-looking map of the wrong thing.
Read size, amount, or a mobility shift
The output is presence, cleavage, ubiquitination, glycosylation, or a co-purifying partner. A mobility shift of spike S2 can be a glycan change rather than a change in abundance; co-detection of two names is not a solved atomic interface.
Treat the band as that species under this blot, not a map
A western answers whether this species changed size or amount under this perturbation. It does not prove localisation or in-vivo dynamics, and proximity catalogues or CRISPR titres sitting in the same index are not blot evidence.
Common misconceptions
A mobility shift of spike S2 means the protein is more abundant.
LYQD mutants altered S2 glycosylation mobility — a size/glycan change — while palmitoylation at cysteine clusters I/II controlled trafficking and particle production. Mobility is not the same as band intensity.
If N's TIR domain binds TMV p50 on a blot, the atomic interface is solved.
Co-expression triggers HR in two days and the TIR domain is critical for association; the paper does not solve a full atomic structure of the N–p50 complex.
Caveolin–cavin co-detection on blots means EHD2 and pacsin 2 are in the same coat complex.
Quantitative composition found caveolins and cavins in one complex that excludes EHD2/pacsin 2. Co-purification defines the coat; it does not place every caveola-associated protein inside that core.
Related
Claim ledger
What the evidence shows
Drawn from 12 studies in this library. Mix labels say which citation roles are present; they are not a strength score. Supports means evidence for a finding; Challenges means evidence against a stated position; Qualifies marks scope.
Caveolar-coat purification used quantitative composition to show caveolins and cavins in one complex that excludes EHD2/pacsin 2, with Cavin 1 a core component and evidence for trimers. Western or blot-style detection of coat subunits supports stoichiometry; it is not a live-cell movie of every tissue's caveolae.
Tagged tobacco N and TMV p50 co-expressed in N. benthamiana associated in the cytoplasm; the TIR domain of N was critical for that association, and co-expression triggered hypersensitive-response cell death in two days. Domain mapping of a physical association is a classic immunoblot/co-IP use, not an atomic structure of the complex.
USP8 limits Smoothened ubiquitination: RNAi and biochemical readouts showed less ubiquitin on Smo, stronger Hedgehog signalling, and Hh-stimulated Smo–USP8 interaction. Ubiquitin blots here are a modification assay tied to trafficking, not a complete ligase identification.
SARS-CoV-2 spike LYQD mutants changed S2 glycosylation mobility, and palmitoylation at cysteine clusters I/II was required for efficient particle production, Golgi/plasma-membrane targeting, and ACE2-mediated fusion (2-bromopalmitate inhibition). Mobility shifts on S2 are a western-style glycosylation readout; much of the work uses spike pseudoviruses rather than full-virus genetics for every assay.
Not every tagged paper is a western-first study. APEX2 maps (22 OMM, 72 ERM proteins) are mass-spectrometry inventories; Wnt5a work uses purified ligand and receptor context to turn β-catenin–TCF signalling on or off; Cas13a plant antiviral work measures GFP and viral accumulation after targeting TuMV regions. Blots may appear as supporting assays, not as the experimental core.
- Mapping the faces of mitochondria and ER
- When does Wnt5a turn β-catenin signaling on or off?
- Can Cas13a fight RNA viruses in plants?
Study Role Design N Population Outcome Mapping the faces of mitochondria and ER Supports Animal / in-vitroAPEX2 proximity labeling of cytosol-facing OMM and ER membranes in living HEK 293T cells SILAC proteomic maps with two replicates per membrane — cell-resource study HEK 293T cells expressing OMM/ERM APEX2 fusions Proteomes of cytosol-facing outer mitochondrial and ER membranes When does Wnt5a turn β-catenin signaling on or off? Supports Animal / in-vitroPurified Wnt5a across receptor contexts testing canonical vs inhibitory outputs Receptor-context cell signaling assays — no single sample N Cells expressing Wnt receptors and responding to purified Wnt5a Context-dependent activation or inhibition of β-catenin–TCF signaling by Wnt5a Can Cas13a fight RNA viruses in plants? Supports Animal / in-vitroProgrammed Cas13a targeting TuMV genomic regions in plants Plant molecular interference assays — no single primary analytic N in stored text Plants infected with Turnip mosaic virus (TuMV) Cas13a-mediated reduction of viral GFP and accumulation
Debates
Tensions and limits
Some items are genuine disagreements on the same question. Others mark different assays, populations, or outcomes.
Westerns here answer different biochemical questions. Caveolar work asks which subunits co-purify; N–p50 asks which domain is required for association and HR; USP8 asks whether Smo is ubiquitinated; spike work asks whether glycan occupancy and palmitoylation change S2 mobility and trafficking. A darker band is not a universal 'more protein' story across those designs.
Westerns here answer different biochemical questions. Caveolar work asks which subunits co-purify; N–p50 asks which domain is required for association and HR; USP8 asks whether Smo is ubiquitinated; spike work asks whether glycan occupancy and palmitoylation change S2 mobility and trafficking. A darker band is not a universal 'more protein' story across those designs.
- What proteins build the caveolar coat?
- Plant TIR domain binds viral elicitor
- How does USP8 turn on Smoothened?
- Spike glycosylation and palmitoylation trafficking
Study Role Design N Population Outcome What proteins build the caveolar coat? Supports Animal / in-vitroBiochemical purification and ultrastructure of caveolar coat proteins Structural/biochemical coat stoichiometry study — no single sample N Caveolar coat complexes (caveolins/cavins) Molecular composition and ultrastructure of the caveolar coat Plant TIR domain binds viral elicitor Supports Animal / in-vitroCo-expression domain mapping of N and TMV p50 in N. benthamiana Plant molecular genetics / protein-association assays — no single sample N Nicotiana benthamiana expressing tagged N and TMV p50 TIR-domain requirement for N–p50 association and HR cell death How does USP8 turn on Smoothened? Supports Animal / in-vitroRNAi screen of Drosophila DUBs in wings and S2 cells focused on USP8–Smo regulation Drosophila genetics and S2 cell assays — no single sample N Drosophila tissues and S2 cells USP8 prevention of Smoothened ubiquitination promoting Hedgehog signaling Spike glycosylation and palmitoylation trafficking Supports Animal / in-vitroMutagenesis and 2BP inhibition of SARS-CoV-2 spike glycosylation/palmitoylation trafficking Cell-based spike trafficking/fusion assays — no single primary analytic N Cells expressing wild-type or mutant SARS-CoV-2 spike Spike intracellular trafficking, packaging, and ACE2-mediated fusion
PaperFren reads this as a limit on how far one study travels — different assays, populations, or outcomes — not a forced fight between papers.
APEX2, Wnt5a, and Cas13a illustrate false-positive or peripheral lexicon hits: proximity proteomics, ligand/receptor signalling, and CRISPR interference on an RNA virus. Citing them as if they were western-blot method papers overstates how the method was used.
APEX2, Wnt5a, and Cas13a illustrate false-positive or peripheral lexicon hits: proximity proteomics, ligand/receptor signalling, and CRISPR interference on an RNA virus. Citing them as if they were western-blot method papers overstates how the method was used.
- Mapping the faces of mitochondria and ER
- When does Wnt5a turn β-catenin signaling on or off?
- Can Cas13a fight RNA viruses in plants?
Study Role Design N Population Outcome Mapping the faces of mitochondria and ER Supports Animal / in-vitroAPEX2 proximity labeling of cytosol-facing OMM and ER membranes in living HEK 293T cells SILAC proteomic maps with two replicates per membrane — cell-resource study HEK 293T cells expressing OMM/ERM APEX2 fusions Proteomes of cytosol-facing outer mitochondrial and ER membranes When does Wnt5a turn β-catenin signaling on or off? Supports Animal / in-vitroPurified Wnt5a across receptor contexts testing canonical vs inhibitory outputs Receptor-context cell signaling assays — no single sample N Cells expressing Wnt receptors and responding to purified Wnt5a Context-dependent activation or inhibition of β-catenin–TCF signaling by Wnt5a Can Cas13a fight RNA viruses in plants? Supports Animal / in-vitroProgrammed Cas13a targeting TuMV genomic regions in plants Plant molecular interference assays — no single primary analytic N in stored text Plants infected with Turnip mosaic virus (TuMV) Cas13a-mediated reduction of viral GFP and accumulation
PaperFren reads this as a limit on how far one study travels — different assays, populations, or outcomes — not a forced fight between papers.
Timeline
How understanding moved
Study years are when the paper was published. Evidence edits are dated changes to this page's claims. Explanations are when PaperFren added a Discovery — not a claim that the science happened that day.
2026
Concept page published
Western Blot
Change log
What changed
Dated edits to this page's evidence: studies added or removed from a claim, claims added or withdrawn, and new explanations tagged here. Coverage edits are distinct from reviewed research changes and corrections. Dates record PaperFren updates; study publication dates appear in the timeline.
- Coverage updated · Concept page published
Papers
12 studies in this library bear on Western Blot, ordered by citations.
- When does Wnt5a turn β-catenin signaling on or off?
Purified Wnt5a can activate or inhibit β-catenin–TCF signaling depending on which Frizzled/receptor context is present.
- Can Cas13a fight RNA viruses in plants?
CRISPR/Cas13a interferes with TuMV in plants, cutting viral RNA and reducing reporter/virus accumulation.
- Mapping the faces of mitochondria and ER
APEX2 proximity biotinylation yields high-quality proteomic maps of cytosol-facing outer mitochondrial and ER membranes in living human cells.
- How interferon throttles sterol synthesis in antiviral defense
Type I interferon couples viral sensing to down-regulation of the sterol pathway’s mevalonate–isoprenoid arm, limiting viral growth.
- Plant TIR domain binds viral elicitor
Tobacco N protein’s TIR domain is critical for association with TMV p50 elicitor.
- How do E. coli vesicles talk to gut epithelium?
Outer membrane vesicles from probiotic and commensal E. coli deliver ligands that activate NOD1-mediated immune responses in intestinal epithelial cells.
- What proteins build the caveolar coat?
Caveolins and cavins purify as a caveolar coat complex with Cavin 1 as a core trimeric component.
- How does USP8 turn on Smoothened?
USP8 deubiquitinates Smoothened, promoting Hh-dependent cell-surface accumulation and signaling while limiting early-endosome localization.
Show 4 more studiesShow fewer studies
- Histone acylations respond to plant stress
Rice histone butyrylation and crotonylation mark active chromatin and shift under starvation/submergence.
- How does ZFP36 reshape metabolism after growth signals?
Growth factors induce ZFP36, which binds and decays metabolic enzyme/transporter mRNAs—especially Eno2—tuning glycolytic metabolism.
- Folliculin tunes mTORC1 toward TFE3
FLCN–RagC signaling makes mTORC1 substrate-selective for TFE3 phosphorylation under amino-acid cues.
- Spike glycosylation and palmitoylation trafficking
Glycosylation motifs and cysteine palmitoylation steer SARS-CoV-2 spike maturation, Golgi trafficking, and fusion.
Compare studies
Select 2–10 studies. Design and N are labels, not a ranking.
Nothing selected yet.
Questions
What is still open
Westerns here answer different biochemical questions. Caveolar work asks which subunits co-purify; N–p50 asks which domain is required for association and HR; USP8 asks whether Smo is ubiquitinated; spike work asks whether glycan occupancy and palmitoylation change S2 mobility and trafficking. A darker band is not a universal 'more protein' story across those designs.
APEX2, Wnt5a, and Cas13a illustrate false-positive or peripheral lexicon hits: proximity proteomics, ligand/receptor signalling, and CRISPR interference on an RNA virus. Citing them as if they were western-blot method papers overstates how the method was used.
Ask PaperFren about Western Blot
Study this conceptflashcards and short-answer questions
Why can a glycosylation mobility shift of SARS-CoV-2 S2 and a ubiquitin smear on Smoothened both be 'western' results while answering different trafficking questions?
S2 mobility reports glycan occupancy after LYQD mutation; Smo ubiquitin blots report a post-translational mark that USP8 removes so Smo can reach the surface. Both are antibody/size readouts, but one is processing and the other is a degradative/trafficking modification.
N and p50 are cytoplasmic and HR appears in two days. What does a TIR-domain association blot add that the HR phenotype alone does not?
HR shows a death outcome of co-expression. The association assay shows a physical N–p50 interaction that requires the TIR domain, which HR timing alone cannot localise to a domain.
Caveolar composition excludes EHD2/pacsin 2. If a student sees those proteins on caveolae by microscopy, how do you reconcile the blot-based complex?
Microscopy can place proteins at the same structure; co-purification of a coat complex is stricter. Cavin 1 is core and trimeric in the biochemical model; EHD2/pacsin 2 can be caveola-associated without sitting in the caveolin–cavin coat.
Cas13a targeting of TuMV reduced GFP and viral accumulation. Why is that a weak example if you are studying western blot as a method?
The headline method is CRISPR/Cas13a interference on an RNA virus, with guide-region effects on GFP and viral load. Any blot is a supporting accumulation readout, not a protein-modification or co-IP study.