hopx Antibody

What is HOPX and why is it important in research?

HOPX (Homeodomain-only protein) is an atypical homeodomain protein that lacks certain conserved residues required for DNA binding. It functions as a regulator of cell differentiation in multiple tissues and has emerged as a crucial marker of specific developmental and differentiation potentials . HOPX is required to modulate cardiac growth and development, acting via its interaction with Serum Response Factor (SRF) to modulate the expression of SRF-dependent cardiac-specific genes . It has also been identified as a potential tumor suppressor in multiple cancers and plays significant roles in immune cell function . Its diverse biological roles make it an important target for antibody-based research across multiple fields.

Different HOPX antibodies show varying reactivity patterns:

When selecting an antibody for cross-species applications, verify the reactivity in the specific intended application, as reactivity may vary between applications even for the same antibody .

How should I optimize HOPX antibody dilution for different applications?

Optimization of HOPX antibody dilution depends on the specific application, sample type, and detection method:

• Western Blot: Begin with manufacturer-recommended dilutions (typically 1:500-1:3000) . Perform a dilution series to determine optimal signal-to-noise ratio.

• Immunohistochemistry: Start with recommended dilutions (typically 1:500-1:2000) . For human placenta or mouse lung tissue, consider antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0 as specifically noted for HOPX antibodies .

• Flow Cytometry: Titration is essential as HOPX expression varies significantly between cell populations . Use a reporter system like Hopx-GFP when possible for accurate detection .

• General Approach: The optimal antibody concentration varies by species and antibody affinity. For each product and application, optimize the antibody titer methodically using positive and negative controls .

Sample-dependent optimization may be necessary as HOPX expression varies significantly between tissues and cell types .

What is the expected molecular weight of HOPX in Western blot applications?

HOPX protein detection can show variability in molecular weight:

• Calculated molecular weight: 8 kDa (73 amino acids)

• Observed molecular weight: Typically 8-12 kDa on Western blots

• Anomalous observations: Some antibodies report observation at significantly higher weights (e.g., 68 kDa) , which may represent specific isoforms, post-translational modifications, or protein complexes.

When performing Western blot analysis for HOPX, include positive controls (mouse lung tissue, mouse small intestine tissue, rat lung tissue) that have been validated for the specific antibody being used .

What are the optimal storage conditions for HOPX antibodies?

For maximum stability and performance of HOPX antibodies:

• Short-term storage: 4°C for up to two weeks is generally recommended for immediate use .

• Long-term storage:

  • Store at -20°C

  • Aliquot into small volumes (at least 20 μl) to avoid freeze-thaw cycles

  • For some antibodies, addition of 50% glycerol in the storage buffer enhances stability at -20°C

• Handling precautions:

  • Avoid repeated freeze-thaw cycles

  • Note that many antibodies contain preservatives like sodium azide (0.02-0.09%), which should be handled with appropriate safety precautions

Antibody stability at 4°C is highly variable between products; therefore, long-term storage at -20°C with proper aliquoting is preferred for most HOPX antibodies .

What is the pattern of HOPX expression in immune cell populations?

HOPX shows distinct expression patterns across immune cell populations:

• T cells:

  • CD8+ T cells: Majority of splenic and lymph node CD8+ T cells express HOPX

  • CD4+ T cells: Only a relatively small portion express HOPX, including specific pre-effector subsets

  • Regulatory T cells (Tregs): HOPX expression is found in peripherally induced Treg cells, where it regulates AP-1 transcription factors and IL-2 production

• B cells:

  • Preferential expression in naïve B cells

  • Contrary to previous reports, HOPX expression is not prominent in IgD-negative or CD24-high B cells

• Other immune cells:

  • Natural killer (NK) cells, NKT cells, and some myeloid cells express HOPX

  • Conventional dendritic cells and eosinophils show minimal HOPX expression

Single-cell RNA sequencing (scRNA-seq) and flow cytometric analysis using Hopx-GFP reporter mice have been crucial in characterizing HOPX expression across immune cell populations .

How does HOPX function in T cell biology and what methods are used to study this?

HOPX plays important regulatory roles in T cell biology:

• Functional roles:

  • In Treg cells: Regulates AP-1 transcription factors and IL-2 production, affecting Treg fitness under inflammatory conditions

  • Marks specific pre-effector differentiation potentials in CD4+ T cells following antigen-specific activation

  • May influence T cell responses in melanoma and other cancers

• Methodological approaches:

  • Flow cytometry with Hopx-GFP reporter mice: Enables detection of Hopx expression when reliable flow cytometry antibodies for murine Hopx are unavailable

  • Single-cell RNA sequencing: Provides comprehensive expression patterns in heterogeneous immune cell populations

  • Functional assays: Assessment of cytokine production, cell proliferation, and activation markers after manipulation of HOPX expression

• Correlation with immune infiltration:

  • In cutaneous melanoma, HOPX expression correlates with immune processes and T cell enrichment, suggesting a role as an immune checkpoint

  • GO/KEGG analysis shows HOPX functions primarily relate to cytokine-cytokine receptor interaction, cell adhesion molecules, and T-cell activation

Researchers investigating HOPX in immune contexts should consider both genetic approaches (reporter systems, knockout models) and antibody-based detection methods depending on the specific research question .

How is HOPX involved in tumor suppression, and what experimental approaches reveal this function?

HOPX demonstrates tumor-suppressive properties across several cancer types, particularly in cutaneous melanoma (SKCM):

• Expression patterns in cancer:

  • HOPX expression is significantly lower in SKCM cells compared to normal cells (p < 0.001)

  • Patients with high HOPX expression have better prognosis (p < 0.01)

  • HOPX shows good diagnostic efficacy as a biomarker (AUC = 0.744)

• Functional effects in cancer cells:

  • Inhibits proliferation, migration, and invasion of melanoma cells

  • Promotes apoptosis and S-phase cell cycle arrest

  • May enhance sensitivity to clinical chemotherapeutic agents

• Experimental approaches:

  • Bioinformatics analysis using TCGA, GEO, and HPA databases to assess expression levels and clinical correlations

  • In vitro cellular assays with HOPX overexpression or knockdown to assess functional effects

  • Transcriptome sequencing to analyze HOPX interactions with related genes

  • Correlation with immune infiltration through GO/KEGG analysis, GSVA, and single-cell sequencing

HOPX functions as a biomarker for both diagnosis and prognosis in cancer, with mechanistic studies suggesting its role in regulating cell proliferation and apoptosis pathways .

What role does HOPX play in alveolar epithelial cells during injury and repair?

HOPX demonstrates dynamic expression changes in alveolar epithelial cells that reflect lung injury and repair processes:

• Expression dynamics:

  • HOPX expression increases in cultured alveolar type II (ATII) cells over time, correlating with decreased proSP-C (an ATII marker)

  • HOPX expression increases in alveolar epithelial cells (AECs) from bleomycin-instilled mouse lungs (modeling pulmonary fibrosis)

  • Flow cytometry analysis shows significant decrease of HOPX-/proSP-C+ cells and increase of HOPX+/proSP-C+ cells in bleomycin-treated primary mouse ATII cells

• Functional significance:

  • siRNA-based knockdown of Hopx:

    • Suppresses ATII to ATI trans-differentiation

    • Activates cellular proliferation in vitro

    • Increases Mki67 and Sftpc expression

    • Enhances metabolic activity in alveolar epithelial cells

  • Ki67+ proliferating cells are significantly less common within HOPX+ cell fractions compared to HOPX- fractions in bleomycin-injured lungs

• Methodological approaches:

  • Flow cytometry-based methods for evaluating HOPX-expressing cells in the lung

  • siRNA knockdown in alveolar epithelial cell lines

  • EdU-based proliferation assays

  • Scratch wound healing assays

  • Immunofluorescence microscopy of lung tissue sections

These findings suggest HOPX is involved in suppressing alveolar epithelial cell proliferation during injury response and may regulate differentiation processes during repair .

What technical challenges exist in detecting and studying specific HOPX isoforms?

Studying HOPX isoforms presents several technical challenges:

• Isoform complexity:

  • At least three isoforms of HOPX are known to exist

  • Different antibodies may detect specific or all isoforms depending on the epitope location

• Detection challenges:

  • Molecular weight variability (observed 8-12 kDa vs. calculated 8 kDa)

  • Some antibodies report much higher apparent molecular weights (e.g., 68 kDa) , potentially representing complexes or artifacts

  • Limited availability of isoform-specific antibodies

• Methodological approaches:

  • Epitope mapping: Use antibodies targeting different regions of HOPX (N-terminal vs. C-terminal)

  • Validation with recombinant protein standards expressing specific isoforms

  • Complementary genetic approaches:

    • Hopx-GFP reporter systems for functional studies when antibody detection is challenging

    • siRNA targeting all or specific isoforms

• Species-specific considerations:

  • "A reliable flow cytometry adaptable antibody specific to murine Hopx is not available" , necessitating reporter systems for mouse studies

  • Human HOPX antibodies tend to have more validation data available

When studying HOPX isoforms, researchers should carefully select antibodies based on the epitope location and validate detection specificity, potentially using complementary genetic approaches to overcome antibody limitations .

What are the most common issues when using HOPX antibodies and how can they be addressed?

Common challenges with HOPX antibodies include:

• Variable signal intensity:

  • Issue: Inconsistent Western blot or IHC signals between experiments

  • Solution: Standardize protein loading, optimize antibody concentration (1:500-1:3000 for WB; 1:500-1:2000 for IHC) , and ensure proper antigen retrieval (TE buffer pH 9.0 or citrate buffer pH 6.0 specifically recommended for HOPX)

• Background staining:

  • Issue: High background in immunohistochemistry or immunofluorescence

  • Solution: Increase blocking time, optimize antibody dilution, and consider using antigen affinity-purified antibodies

• Molecular weight discrepancies:

  • Issue: Detection at unexpected molecular weights (expected: 8-12 kDa)

  • Solution: Include positive controls (mouse lung tissue, mouse small intestine tissue, rat lung tissue) and consider alternative antibodies targeting different epitopes

• Reactivity differences:

  • Issue: Inconsistent results across species

  • Solution: Select antibodies with validated cross-species reactivity and perform preliminary validation in your specific species/tissue

• Detection in mouse tissues:

  • Issue: Limited availability of reliable flow cytometry antibodies for murine Hopx

  • Solution: Use Hopx-GFP reporter mice when studying mouse tissues/cells

For optimal results, researchers should titrate each antibody for their specific application and sample type, as recommended dilutions provide only starting points for optimization .

How should I design experiments to study HOPX function in cells and tissues?

Design robust experiments to study HOPX function with these methodological considerations:

• Expression analysis approaches:

  • Protein level: Western blot (1:500-1:3000 dilution) , immunohistochemistry (1:500-1:2000) , immunofluorescence

  • Transcript level: qRT-PCR with appropriate reference genes

  • Single-cell level: Flow cytometry or single-cell RNA sequencing

  • Spatial distribution: Immunofluorescence microscopy with co-staining for lineage markers

• Functional studies:

  • Loss-of-function: siRNA knockdown , CRISPR-Cas9 knockout

  • Gain-of-function: Overexpression systems

  • Readouts:

    • Proliferation: EdU incorporation, Ki67 staining, scratch assays

    • Differentiation: Lineage marker expression (e.g., proSP-C for ATII cells)

    • Cell death: Apoptosis assays

• Controls and validation:

  • Positive tissue controls: Mouse lung tissue, mouse small intestine tissue, rat lung tissue for HOPX antibodies

  • Knockdown/knockout validation: Western blot and qRT-PCR verification

  • Multiple antibodies: Use antibodies targeting different epitopes to confirm specificity

• Context-specific considerations:

  • Immune cells: Consider heterogeneity within populations; use sorting or single-cell approaches

  • Cancer studies: Compare paired normal/tumor samples; correlate with clinical parameters

  • Lung injury models: Compare baseline and injury conditions; track temporal changes

Well-designed HOPX functional studies should incorporate multiple complementary techniques and appropriate controls to ensure robust and reproducible findings .

What emerging techniques are advancing HOPX antibody-based research?

Several innovative approaches are enhancing HOPX antibody applications:

• High-resolution imaging technologies:

  • Super-resolution microscopy for detailed subcellular localization

  • Multiplexed immunofluorescence for simultaneous detection of HOPX with multiple markers

  • Spatial transcriptomics combined with protein detection to correlate HOPX protein and mRNA levels in tissue context

• Single-cell analysis:

  • Mass cytometry (CyTOF) for high-dimensional analysis of HOPX expression across cell populations

  • Integrated single-cell RNA-seq and antibody-based protein detection (CITE-seq) to correlate transcriptional and protein expression

  • Advanced flow cytometry with Hopx reporter systems for functional studies

• Proximity labeling approaches:

  • BioID or APEX2-based proximity labeling to identify HOPX interaction partners

  • PLA (Proximity Ligation Assay) to detect specific HOPX protein-protein interactions in situ

• Translational applications:

  • Development of HOPX antibodies with improved specificity for detecting particular isoforms

  • Expansion of validated applications to additional species and tissues

  • Integration of HOPX detection in clinical diagnostic panels based on its role as a prognostic biomarker

These emerging methodologies promise to provide deeper insights into HOPX biology and function across different cellular contexts .

What are the current knowledge gaps in HOPX biology that antibody-based research could address?

Several important knowledge gaps in HOPX biology could be addressed through antibody-based research:

• Isoform-specific functions:

  • Development of isoform-specific antibodies to distinguish the roles of different HOPX variants

  • Characterization of isoform expression patterns across tissues and disease states

  • Investigation of isoform-specific protein interaction networks

• Mechanistic understanding:

  • Elucidation of HOPX's role in transcriptional regulation despite lacking DNA binding capability

  • Characterization of HOPX protein complexes using co-immunoprecipitation with specific antibodies

  • Investigation of post-translational modifications affecting HOPX function

• Clinical applications:

  • Validation of HOPX as a diagnostic/prognostic biomarker across multiple cancer types

  • Exploration of HOPX expression as a predictor of treatment response

  • Development of standardized HOPX immunohistochemistry protocols for clinical application

• Therapeutic implications:

  • Understanding how modulation of HOPX expression affects tumor suppression mechanisms

  • Investigation of HOPX's role in immune checkpoint regulation in cancer immunotherapy

  • Exploration of HOPX as a target for promoting proper tissue regeneration after injury

Antibody-based approaches, combined with genetic and functional studies, will be essential to address these knowledge gaps and advance our understanding of HOPX biology .