hopP Antibody

What is HOP protein and why is it significant for antibody development?

HOP (Hsp70-Hsp90 Organizing Protein), also known as STIP1 or p60, is a ~60kDa protein that serves as a critical intermediate component for the efficient maturation of steroid receptor complexes. It functions by recruiting Hsp90 to Hsp70-containing complexes . The protein contains three tetratricopeptide repeat (TPR) domains: TPR1, TPR2a, and TPR2b, which are important for its function .

The significance of HOP for antibody development stems from its conserved structure across multiple species and its involvement in essential cellular processes, making it an important target for studying chaperone-mediated protein folding and stress responses. Anti-HOP antibodies allow researchers to investigate these pathways in various experimental contexts.

How do different types of HOP antibodies (monoclonal vs. polyclonal) compare in research applications?

What experimental applications are most suitable for HOP antibodies?

HOP antibodies have demonstrated utility in multiple research applications:

• Western Blotting (WB): HOP antibodies reliably detect a band of ~60kDa, with recommended dilutions typically around 1:1,000 for colorimetric detection .

• Immunohistochemistry (IHC): Both paraffin-embedded and frozen sections can be analyzed, with HOP antibodies recommended by organizations like the Human Protein Atlas for IHC applications (e.g., Ensembl No. ENSG00000168439) .

• Immunoprecipitation (IP): HOP antibodies effectively precipitate HOP protein and its complexes with Hsp70/Hsp90, enabling studies of chaperone interactions .

• Flow Cytometry: Though less common, certain HOP antibodies can be used in flow cytometry applications when properly validated.

Experimental validation shows that applications must be optimized for each specific antibody. For instance, the DS14F5 clone has been validated for WB, IHC, and IP applications across multiple species including human, mouse, rat, and others .

How can I optimize HOP antibody performance for studying protein-protein interactions?

Optimizing HOP antibody performance for protein-protein interaction studies requires careful consideration of several factors:

• Selection of antibody epitope: Choose antibodies targeting epitopes that don't interfere with the protein-protein interaction domains of interest. For HOP, avoid antibodies targeting the TPR domains if you're studying Hsp70-Hsp90 interactions .

• Cross-linking protocols: For co-immunoprecipitation experiments:

  • Use mild detergents (0.5% NP-40 or 1% Triton X-100)

  • Include protease inhibitors to prevent degradation

  • Consider reversible cross-linkers like DSP (dithiobis[succinimidylpropionate]) to stabilize transient interactions

• Buffer optimization: For studying HOP-chaperone complexes:

  • Include 20mM sodium molybdate to stabilize complexes

  • Maintain ATP at physiological concentrations (1-5mM)

  • Use buffers that maintain native protein conformation (HEPES or phosphate at pH 7.2-7.4)

• Validation controls: Always include:

  • Isotype controls for monoclonal antibodies

  • Pre-immune serum controls for polyclonal antibodies

  • Blocking peptide competition assays to confirm specificity

• Advanced techniques: Consider proximity ligation assays (PLA) or FRET-based approaches to study HOP interactions in situ with minimal disruption to cellular environments.

What methodological approaches can help distinguish between free HOP and HOP-chaperone complexes?

Distinguishing between free HOP and HOP-chaperone complexes requires specialized methodological approaches:

• Size-based separation techniques:

  • Native PAGE followed by Western blotting can separate free HOP (~60kDa) from complexes (>200kDa)

  • Size exclusion chromatography prior to immunodetection

  • Sucrose gradient ultracentrifugation to fractionate complexes based on size

• Sequential immunoprecipitation:

  • First IP with anti-Hsp70 or anti-Hsp90 antibodies

  • Then probe for co-precipitated HOP

  • Compare with direct HOP immunoprecipitation to determine percentage of complexed HOP

• Proximity-based detection methods:

  • In situ proximity ligation assay (PLA) to visualize HOP-chaperone interactions in fixed cells

  • FRET or BRET between labeled HOP and chaperone proteins in living cells

• Chemical cross-linking coupled with mass spectrometry:

  • Use mild cross-linkers to stabilize complexes

  • Digest and analyze by MS to identify interaction sites

  • Compare with non-cross-linked samples to identify specific vs. non-specific interactions

• Fluorescence correlation spectroscopy (FCS):

  • Label HOP antibodies with fluorophores

  • Measure diffusion coefficients to distinguish between free and complexed HOP

These approaches can be combined for more comprehensive analysis of HOP-chaperone dynamics in different cellular contexts.

How can HOP antibodies be used to investigate stress response pathways?

HOP antibodies are valuable tools for investigating stress response pathways due to HOP's central role in chaperone networks. Methodological approaches include:

• Stress induction experimental design:

  • Heat shock (42°C for 1-2 hours)

  • Oxidative stress (H₂O₂ treatment)

  • ER stress inducers (tunicamycin, thapsigargin)

  • Hypoxia (1-2% O₂)

  • Proteasome inhibitors (MG132, bortezomib)

• Subcellular fractionation and immunodetection:

  • Track HOP relocalization between cytoplasm and nucleus during stress

  • Use HOP antibodies for immunofluorescence to visualize redistribution

  • Compare patterns with other chaperone proteins (Hsp70, Hsp90)

• Immunoprecipitation under stress conditions:

  • Compare HOP-associated proteins before and after stress

  • Identify stress-specific clients using co-IP followed by mass spectrometry

  • Use phospho-specific antibodies to detect stress-induced post-translational modifications

• Chromatin immunoprecipitation (ChIP):

  • Some research suggests HOP may associate with chromatin during stress

  • Use HOP antibodies for ChIP to identify potential DNA binding sites

  • Combine with RNA-seq to correlate with stress-induced gene expression changes

• Proteomic screening approaches:

  • Use HOP antibodies for immunoaffinity purification

  • Couple with mass spectrometry to identify stress-dependent interaction partners

  • Validate key interactions with reciprocal co-IP and immunofluorescence colocalization

When designing these experiments, researchers should include appropriate controls for stress induction and consider the kinetics of the stress response, as HOP-chaperone interactions may change dynamically over time.

What are common challenges in HOP antibody specificity and how can they be addressed?

When troubleshooting specificity issues, it's important to note that antibodies like DS14F5 have been validated for cross-reactivity with multiple species including bovine, chicken, dog, guinea pig, hamster, human, mink, monkey, mouse, porcine, rabbit, rat, sheep, and Xenopus , but each specific application may require optimization.

How can I properly validate a HOP antibody for my specific experimental system?

A comprehensive validation strategy for HOP antibodies includes:

• Primary validation techniques:

  • Western blot analysis: Confirm band size (~60kDa) in multiple cell types/tissues

  • siRNA/shRNA knockdown: Show reduced signal with HOP depletion

  • Knockout controls: Where available, use CRISPR-generated HOP knockout cells

  • Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

  • Recombinant protein analysis: Test against purified HOP protein

• Application-specific validation:

  • For IHC/ICC: Compare staining patterns with multiple antibodies targeting different HOP epitopes

  • For IP: Confirm pulled-down protein by mass spectrometry or Western blot

  • For flow cytometry: Use parallel techniques (e.g., ICC) to confirm expression patterns

• Species cross-reactivity validation:

  • Test antibody against lysates from multiple species

  • Align epitope sequences across species to predict reactivity

  • Consider species-specific positive controls

• Documentation of validation experiments:

  • Record all experimental conditions (buffers, dilutions, incubation times)

  • Include all controls in publication materials

  • Report batch/lot numbers of antibodies used

For HOP antibodies specifically, validation should include assessment of binding under conditions that maintain or disrupt HOP-chaperone complexes, as this can affect epitope accessibility.

What are the critical parameters for optimizing HOP antibodies in immunohistochemistry applications?

When optimizing IHC for HOP antibodies, researchers should note that HOP predominantly localizes to the cytoplasm but can translocate to the nucleus under certain stress conditions, so both compartments should be evaluated for staining.

How can epitope-specific HOP antibodies be rationally designed for targeting specific protein domains?

Rational design of epitope-specific HOP antibodies can be achieved using several methodological approaches:

• Computational epitope prediction:

  • Analyze HOP sequence using Hopp-Woods hydrophilicity scale to identify exposed, hydrophilic regions likely to be antigenic

  • Use structural prediction algorithms to identify surface-exposed regions

  • Target regions with high predicted antigenicity scores but avoid highly conserved domains if species specificity is desired

• Structure-guided epitope selection:

  • Target distinct functional domains within HOP (TPR1, TPR2a, TPR2b) for domain-specific antibodies

  • Avoid regions involved in protein-protein interactions if studying complex formation

  • Select regions with conformational stability to improve antibody performance

• Complementary peptide design approach:

  • Design synthetic peptides complementary to specific HOP epitopes

  • Graft these peptides onto antibody scaffolds, particularly in CDR regions

  • This approach allows precise targeting of specific disordered regions or epitopes

• Validation of domain specificity:

  • Generate truncated HOP constructs expressing specific domains

  • Test antibody binding to each domain to confirm specificity

  • Perform competition assays with domain-specific peptides

• Applications of domain-specific antibodies:

  • TPR1-specific antibodies: Study Hsp70 interactions

  • TPR2a-specific antibodies: Investigate Hsp90 binding

  • C-terminal-specific antibodies: Examine dimerization and client interactions

The rational design method described in search result can be particularly valuable for HOP, as it enables researchers to obtain antibodies targeting specific epitopes within disordered regions of the protein, which may be less immunogenic by traditional methods.

What are the methodological considerations for using HOP antibodies in multiplexed imaging systems?

Implementing HOP antibodies in multiplexed imaging requires careful planning and optimization:

• Antibody compatibility assessment:

  • Test for cross-reactivity between multiple primary antibodies

  • Ensure secondary antibodies don't cross-react with non-target primaries

  • Consider using directly conjugated primary antibodies to avoid secondary antibody issues

• Fluorophore selection for multiplexing:

  • Choose fluorophores with minimal spectral overlap

  • Common combinations with HOP antibodies:

    • HOP (488nm) + Hsp70 (594nm) + Hsp90 (647nm) + DAPI

    • HOP (Cy3) + client proteins (Cy5) + organelle markers (Pacific Blue)

  • Account for tissue autofluorescence when selecting fluorophores

• Sequential staining protocols:

  • Strip-and-reprobe methods:

    • First detection: HOP antibody → secondary → imaging

    • Strip antibodies using glycine (pH 2.5) or SDS buffer

    • Second detection with next antibody

  • Tyramide signal amplification (TSA) for sequential multiplexing:

    • Allows use of same host antibodies for multiple targets

    • Excellent for low-abundance targets

• Advanced multiplexing technologies:

  • Mass cytometry (CyTOF) with metal-tagged antibodies

  • Cyclic immunofluorescence (CyCIF)

  • CODEX multiplexed imaging

• Image acquisition and analysis considerations:

  • Proper channel alignment and registration

  • Spectral unmixing for overlapping fluorophores

  • Quantitative colocalization analysis (Pearson's coefficient, Manders' overlap)

• Controls for multiplexed imaging:

  • Single-stained controls for spectral overlap assessment

  • Isotype controls for each species/isotype of primary antibody

  • Absorption controls with immunizing peptides

These methodological considerations help ensure reliable detection of HOP alongside other proteins of interest in complex biological samples.

How can I use HOP antibodies to study dynamic changes in protein-protein interactions during cellular stress responses?

Studying dynamic HOP-chaperone interactions during stress responses requires specialized approaches:

• Time-course experimental design:

  • Establish baseline HOP interactions in unstressed cells

  • Apply stress stimuli (heat shock, oxidative stress, ER stress)

  • Collect samples at multiple timepoints (5min, 15min, 30min, 1h, 2h, 4h, 8h, 24h)

  • Use HOP antibodies to track changes in complexes over time

• Live-cell imaging approaches:

  • Use cell-permeable fluorescently labeled HOP antibody fragments (Fabs)

  • Combine with labeled Hsp70/Hsp90 to track interactions

  • Perform FRET or BRET analysis to quantify protein proximity

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to measure dynamics

• Pulse-chase immunoprecipitation:

  • Metabolically label proteins (SILAC or AHA)

  • Apply stress and collect at different timepoints

  • Immunoprecipitate with HOP antibodies

  • Analyze co-precipitated proteins by mass spectrometry

  • Quantify changes in interaction partners over time

• Proximity labeling techniques:

  • Generate HOP-BioID or HOP-APEX fusion proteins

  • Activate proximity labeling before/during/after stress

  • Purify biotinylated proteins with streptavidin

  • Identify stress-dependent interaction partners

  • Validate with HOP antibodies by co-IP or immunofluorescence

• Single-molecule imaging:

  • Immobilize HOP antibodies on coverslips

  • Flow cell extracts from stressed/unstressed cells

  • Monitor binding/dissociation of fluorescently labeled chaperones

  • Calculate kinetic parameters of interactions under different conditions

• Analytical considerations:

  • Account for changes in total HOP levels during stress

  • Distinguish between new complex formation and redistribution

  • Consider compartment-specific changes (cytosolic vs. nuclear)

  • Correlate with functional outcomes (client protein folding, degradation)

These methodologies allow researchers to capture the dynamic nature of HOP-mediated chaperone networks during cellular adaptation to stress conditions.

How can HOP antibodies be used in studying autoimmune responses and cross-reactivity with pathogens?

Recent research has identified interesting connections between HOP, autoimmunity, and pathogen interactions:

• Autoantibody detection methodologies:

  • Develop ELISA-based assays using purified HOP protein and patient sera

  • Compare reactivity against different HOP domains to identify immunodominant regions

  • Implement protein microarrays including HOP and related chaperones

• Cross-reactivity analysis with pathogen proteins:

  • Identify pathogen proteins with structural or sequence homology to HOP

  • Test cross-reactivity of anti-HOP antibodies with these pathogen proteins

  • Investigate whether pathogen infection induces anti-HOP autoantibodies

• Applications in infectious disease research:

  • Study potential molecular mimicry between HOP and pathogen proteins

  • Investigate whether HOP antibodies can neutralize certain pathogens through cross-reactivity

  • Research suggests some autoantibodies may inhibit pathogen growth, as observed with Plasmodium falciparum

• Methodological approaches for cross-reactivity studies:

  • Epitope mapping using peptide arrays

  • Competition assays between pathogen proteins and HOP for antibody binding

  • Absorption studies to deplete specific antibody populations

• Potential significance:

  • Certain autoimmune conditions might confer protection against specific infections

  • This phenomenon has been observed in malaria, where autoantibodies have been shown to inhibit Plasmodium falciparum growth

  • HOP antibodies could potentially be part of this protective autoimmune response

These emerging research directions connect HOP antibodies to broader questions about the relationship between autoimmunity and protection against infectious diseases.

What are the latest methodologies for using HOP antibodies in high-throughput screening applications?

High-throughput applications of HOP antibodies are expanding with new technological developments:

• Antibody array technologies:

  • Reverse phase protein arrays (RPPA) incorporating HOP antibodies

  • Multiplex bead-based assays for simultaneous detection of HOP and interacting partners

  • Microfluidic antibody arrays for minimal sample consumption

• Drug screening applications:

  • High-content imaging using HOP antibodies to screen for compounds affecting:

    • HOP localization

    • HOP-chaperone interactions

    • Client protein folding

  • BRET/FRET-based screening assays to identify modulators of HOP interactions

• Automated immunoprecipitation platforms:

  • Magnetic bead-based IP systems for high-throughput analysis

  • Combined with mass spectrometry for interaction partner identification

  • Parallel processing of multiple conditions (drugs, stressors, genetic perturbations)

• Single-cell analysis with HOP antibodies:

  • Mass cytometry for high-dimensional phenotyping

  • Imaging mass cytometry for spatial context

  • Single-cell Western blotting for protein analysis

• CRISPR screening combined with HOP antibody readouts:

  • Genome-wide or focused CRISPR screens

  • Immunofluorescence or flow cytometry with HOP antibodies as readout

  • Identification of genes affecting HOP expression, localization, or interactions

• Methodological considerations:

  • Miniaturization to reduce antibody consumption

  • Automation of staining and washing steps

  • Standardization of positive and negative controls

  • Data management and analysis pipelines for large datasets

These high-throughput approaches enable systematic studies of HOP biology across diverse conditions, cell types, and genetic backgrounds.

How do post-translational modifications of HOP affect antibody recognition and what methodologies can address this challenge?

Post-translational modifications (PTMs) of HOP can significantly impact antibody recognition, requiring specialized methodological approaches:

• Common PTMs affecting HOP:

  • Phosphorylation (multiple serine/threonine sites)

  • Acetylation

  • Ubiquitination

  • SUMOylation

• Impact on antibody recognition:

  • Epitope masking or enhanced exposure

  • Altered protein conformation

  • Modified charge distribution affecting antibody binding

  • Creation of new epitopes (modification-specific)

• PTM-aware antibody selection strategies:

  • Use antibodies targeting regions without known modification sites

  • Employ multiple antibodies targeting different epitopes

  • Consider modification-specific antibodies for particular applications

• Methodological approaches for PTM analysis:

  • Phosphorylation analysis:

    • Lambda phosphatase treatment of samples before antibody application

    • Compare staining patterns before/after treatment

    • Use phospho-specific antibodies alongside total HOP antibodies

  • Other modifications:

    • Deacetylase treatment for acetylation

    • DUB treatment for ubiquitination

    • SENP treatment for SUMOylation

• Advanced PTM detection methods:

  • Phos-tag™ SDS-PAGE to separate phosphorylated forms before Western blotting

  • 2D-PAGE combined with HOP antibody detection

  • Mass spectrometry following HOP immunoprecipitation

  • Proximity ligation assays using PTM-specific antibodies paired with HOP antibodies

• Application-specific considerations:

  • For signaling studies: use phospho-specific antibodies

  • For total HOP quantification: select antibodies unaffected by PTMs

  • For interaction studies: consider how PTMs affect complex formation

Understanding and accounting for PTMs is essential for accurate interpretation of HOP antibody results, particularly in stress response or signaling pathway studies where HOP modification state may change dynamically.