ste4 Antibody

What is STEAP4 and why is it an important research target?

STEAP4 belongs to a family of metalloreductases involved in iron and copper homeostasis and other cellular processes. It functions as a NADPH-dependent ferric-chelate reductase, using NADPH from one side of the membrane to reduce Fe(3+) chelate bound on the other side. The protein mediates sequential transmembrane electron transfer from NADPH to FAD and onto heme, and finally to the Fe(3+) chelate .

STEAP4 plays crucial roles in:

• Systemic metabolic homeostasis

• Integration of inflammatory and metabolic responses

• Regulation of inflammatory cytokines

• Inhibition of anchorage-independent cell proliferation

STEAP4 is highly expressed in placenta, lung, heart, and prostate tissues, and is notably overexpressed in prostate cancer cells compared to normal prostate tissue . As a cell-surface protein that regulates important metabolic and inflammatory pathways, it represents a valuable research target for studying obesity, insulin resistance, inflammation, and cancer biology .

How do I select the appropriate STEAP4 antibody for my specific experimental applications?

When selecting STEAP4 antibodies, consider these methodological factors:

Application compatibility: STEAP4 antibodies are validated for specific applications, such as immunohistochemistry (IHC), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) . Review validation data for your intended application.

Epitope recognition: Consider the epitope recognized by the antibody. Some antibodies target specific regions, such as amino acids 201-250 of human STEAP4 , which may affect detection in complex samples.

Species cross-reactivity: Verify cross-reactivity with your species of interest. Some STEAP4 antibodies demonstrate reactivity across human, rat, and mouse samples , while others might be species-specific.

Clonality considerations:

• Polyclonal antibodies: Better for detecting low-abundance proteins but may exhibit batch-to-batch variability

• Monoclonal antibodies: Provide higher specificity but might miss certain isoforms

Recommended dilution ranges:

Data derived from standard antibody protocols and manufacturer recommendations

How can I verify STEAP4 antibody specificity?

Antibody specificity verification is crucial for reliable research data. Implement these methodological approaches:

• Positive control samples: Use tissues known to highly express STEAP4 (placenta, lung, heart, prostate) .

• Negative control experiments:

  • Omit primary antibody but maintain all other staining steps

  • Use tissues known to lack STEAP4 expression

  • Include isotype control antibodies to assess non-specific binding

• Western blot validation: Confirm that the antibody detects a band of the expected molecular weight.

• Peptide competition assay: Pre-incubate antibody with purified STEAP4 peptide prior to staining; signal should be blocked or significantly reduced.

• siRNA/CRISPR knockdown: Reduce target protein expression and verify corresponding reduction in antibody signal.

• Cross-reactivity assessment: Test against other STEAP family members (STEAP1, STEAP2, STEAP3) to ensure specificity, particularly important as these proteins share structural similarities .

How can I optimize STEAP4 antibody-based assays for studying its role in adipocyte biology?

STEAP4 plays significant roles in adipocyte biology and metabolic regulation. When designing experiments to study these functions:

Experimental design recommendations:

• Cell viability assays: Implement multiple methodologies for robustness:

  • Trypan Blue exclusion for direct cell counting

  • CCK-8 assays for metabolic activity assessment

  • Cell cycle analysis via flow cytometry

• Morphological assessments:

  • Electron microscopy for ultrastructural changes

  • Confocal laser microscopy for protein localization

  • Oil Red O staining for adipogenesis quantification

• Apoptosis detection:

  • Annexin V-FITC labeling

  • Caspase-3 and caspase-8 activity assays

  • qPCR for apoptotic gene expression changes

• Glucose uptake measurement:

  • 2-deoxy-d-[3H]-glucose uptake tests to assess insulin sensitivity

These methodologies have been validated in studies showing that STEAP4 antibodies can inhibit pre-adipocyte proliferation, promote apoptosis in pre-adipocytes, and enhance insulin-stimulated glucose uptake in mature human adipocytes, without affecting adipocyte differentiation .

What are the critical considerations for using STEAP4 antibodies in cancer research applications?

STEAP4 has emerged as a significant target in cancer research, particularly for prostate cancer where it is overexpressed . When designing cancer-focused experiments:

Methodological considerations:

• Tissue microarray analysis: Use STEAP4 antibodies to assess expression patterns across multiple tumor samples and corresponding normal tissues.

• Correlation with clinical parameters: Design studies that correlate STEAP4 expression with:

  • Tumor grade and stage

  • Patient survival outcomes

  • Treatment response

  • Metastatic potential

• Functional studies:

  • Colony formation assays to assess growth effects

  • Migration and invasion assays to evaluate metastatic potential

  • Cell signaling pathway analysis to determine downstream effects

• Combined biomarker analysis: Evaluate STEAP4 expression in conjunction with other STEAP family members (especially STEAP1, STEAP2) as they have distinct expression patterns in prostate cancer:

  • STEAP1, STEAP2, and STEAP4 are typically overexpressed

  • STEAP3 expression is often downregulated

Research significance: Understanding STEAP4's role in cancer may provide insights into novel diagnostic, prognostic, and therapeutic approaches, as STEAP family members are being investigated as biomarkers and therapeutic targets for prostate cancer .

What technical factors influence the compartmental distribution of STEAP4 antibodies in tissue samples?

The compartmental distribution of antibodies in tissues is a critical consideration for accurate interpretation of STEAP4 localization and expression studies. Key technical factors include:

• Tissue architectural considerations:

  • Relative vascular, interstitial, and cellular volumes affect antibody penetration

  • Heterogeneity within tumor samples can lead to uneven antibody distribution

• Antibody biophysical properties:

  • Molecular size impacts tissue penetration

  • Charge and hydrophobicity affect interactions with tissue components

  • Affinity for target influences retention in specific compartments

• Physiological barriers:

  • Vascular permeability varies across tissue types

  • Interstitial pressure can impede antibody diffusion

  • Cell membrane permeability determines intracellular access

• Experimental approaches for accurate assessment:

  • Physiologically based pharmacokinetic modeling

  • Quantitative methods to distinguish between free and bound antibody fractions

  • Spatial composition measurements to determine relative volumes of vascular, interstitial, and cellular compartments

Methodological recommendation: Implement multi-parameter analysis combining immunohistochemistry with quantitative image analysis techniques to accurately assess STEAP4 distribution across cellular compartments.

How can flow cytometry be optimized for STEAP4 detection and quantification?

Flow cytometry offers powerful capabilities for quantitative analysis of STEAP4 expression at the single-cell level. Consider these methodological approaches:

Protocol optimization:

• Cell preparation:

  • For adherent cells expressing STEAP4, use gentle enzymatic dissociation to preserve membrane integrity

  • For tissue samples, optimize tissue disaggregation protocols to maintain surface protein expression

• Fixation considerations:

  • For surface STEAP4 detection: Mild fixation (0.5-1% paraformaldehyde)

  • For intracellular detection: Methanol-based protocols have shown better results than standard formaldehyde fixation

• Antibody incubation:

  • Primary STEAP4 antibody concentration: Begin with 5 μL (0.25 μg) per test (10^5 to 10^8 cells)

  • Optimal incubation time: 30-45 minutes at 4°C for surface staining

  • For intracellular staining: Permeabilization is crucial, with two-step fixation/methanol protocols showing best results

• Quantification approaches:

  • Use Quantum MESH microspheres and fluorochrome to protein ratio for accurate antigen density quantitation

  • MFI (Mean Fluorescence Intensity) can be translated into absolute STEAP4 density measurements using appropriate calibration standards

• Controls:

  • FMO (Fluorescence Minus One) controls to account for spectral overlap

  • Isotype controls to assess non-specific binding

  • Positive controls using cell lines with known STEAP4 expression levels

What are the methodological approaches for studying STEAP4 protein interactions using antibody-based techniques?

Understanding STEAP4's protein interactions is critical for elucidating its role in cellular signaling and function. Consider these methodological approaches:

Co-immunoprecipitation protocol:

• Sample preparation:

  • Culture cells to early log phase

  • Apply appropriate treatments (e.g., cytokine stimulation) to induce interactions

  • Harvest by centrifugation and resuspend in lysis buffer containing:

    • 50 mM NaPO₄

    • Protease inhibitor cocktail

    • Appropriate detergent (e.g., Triton X-100, 0.5-1%)

• Immunoprecipitation procedure:

  • Incubate lysates with anti-STEAP4 antibodies (1:1000 dilution)

  • Add protein A/G beads and incubate with rotation (4°C, 2-4 hours)

  • Wash beads 4-5 times with lysis buffer

  • Elute bound proteins with SDS-PAGE loading buffer

• Detection methods:

  • Resolve by SDS-PAGE (7.5-10% gels optimal for STEAP4)

  • Transfer to nitrocellulose membrane

  • Probe with specific antibodies against suspected interaction partners

  • Visualize using enhanced chemiluminescence detection of HRP-conjugated secondary antibodies

• Controls and validation:

  • Input controls (5-10% of lysate)

  • IgG isotype control immunoprecipitations

  • Reciprocal co-IPs (immunoprecipitate interaction partner, detect STEAP4)

  • STEAP4 knockdown/knockout controls

This approach has been successfully implemented in studies examining protein-protein interactions, providing valuable insights into functional relationships between proteins .

How should I interpret contradictory STEAP4 antibody staining patterns across different sample types?

Contradictory staining patterns are common challenges in antibody-based research. Consider these methodological approaches for resolution:

• Technical variation analysis:

  • Antibody batch differences may cause variable staining

  • Fixation conditions impact epitope accessibility

  • Antigen retrieval methods affect detection sensitivity

• Biological variation assessment:

  • STEAP4 expression varies significantly across tissues (high in placenta, lung, heart, prostate)

  • Post-translational modifications may mask epitopes

  • STEAP4 isoforms may show differential antibody recognition

• Epitope consideration:

  • Some STEAP4 antibodies target specific regions (e.g., amino acids 201-250)

  • Conformational epitopes may be disrupted in certain preparations

  • Protein-protein interactions may block epitope access

• Methodological resolution approach:

  • Implement multiple antibodies targeting different STEAP4 epitopes

  • Correlate antibody staining with mRNA expression data

  • Use orthogonal detection methods (e.g., mass spectrometry)

  • Consider native vs. denatured protein detection methods

What quantitative standards should be applied when measuring STEAP4 expression levels?

Accurate quantitation is essential for reliable STEAP4 research. Consider these methodological standards:

• Absolute quantification approaches:

  • Use purified recombinant STEAP4 protein as quantitative standard

  • Implement calibrated flow cytometry with Quantum MESH microspheres

  • Apply quantitative mass spectrometry with isotope-labeled standards

• Relative quantification considerations:

  • Select appropriate housekeeping controls based on experimental context

  • For Western blot quantitation, Pgk1 has been validated as a stable reference

  • For qPCR, validate reference gene stability across experimental conditions

• Dynamic range assessment:

  • STEAP4 abundance varies significantly across cell types

  • In expression studies, STEAP4 protein abundance has been estimated at approximately 2,000-5,000 molecules per cell in relevant systems

  • Ensure detection methods accommodate this expected range

• Interpretation framework:

  • Consider fold-change relative to appropriate controls

  • Establish thresholds for biological significance based on functional assays

  • Account for tissue-specific baseline expression levels when interpreting changes

How can I troubleshoot non-specific binding issues with STEAP4 antibodies?

Non-specific binding can compromise research findings. Address these issues through:

• Optimization strategies:

  • Titrate antibody concentration (start with manufacturer recommendations, then optimize)

  • Increase washing duration and stringency

  • Add blocking agents (5% BSA, normal serum from secondary antibody species)

  • Reduce incubation temperature (4°C often reduces non-specific interactions)

• Buffer optimization:

  • Adjust salt concentration to modify stringency

  • Add mild detergents (0.05-0.1% Tween-20)

  • Include carrier proteins to reduce non-specific binding

  • Consider specialized blocking buffers for problematic samples

• Validation approaches:

  • Peptide competition assays to confirm specificity

  • STEAP4 knockout/knockdown controls

  • Secondary-only controls to assess non-specific secondary antibody binding

  • Isotype controls to evaluate Fc-mediated binding

• Alternative detection strategies:

  • Consider directly conjugated primary antibodies to eliminate secondary antibody issues

  • Implement amplification systems with enhanced specificity (e.g., TSA)

  • Test alternative detection systems (e.g., quantum dots, fluorescent proteins)

How can STEAP4 antibodies be utilized in bispecific antibody development for therapeutic applications?

Bispecific antibodies (BsAbs) represent an emerging frontier in STEAP4-directed therapeutics. Consider these methodological approaches:

• Platform selection considerations:

  • IgG-[L]-scFv platform has been effectively used for STEAP1 BsAbs

  • 2+2 format (2 tumor binding domains + 2 CD3 binding domains) shows greater potency than 2+1 format

• Experimental characterization requirements:

  • Epitope mapping to identify optimal binding regions

  • Species cross-reactivity testing (human, dog, non-human primate)

  • Cell line binding assessments

  • In vitro cytotoxicity evaluations

• Efficacy assessment approaches:

  • Bioluminescence quantification of tumor-infiltrating lymphocyte recruitment

  • Xenograft model testing (both cell line-derived and patient-derived)

  • Comparative analysis against alternative antibody platforms

• Target validation considerations:

  • STEAP family members (particularly STEAP1 and STEAP4) show promise as therapeutic targets

  • Limited expression in normal tissues (except secretory tissues) makes them suitable for antibody-based therapies

  • Correlation between overexpression and disease progression supports therapeutic potential

What methodological approaches are recommended for studying STEAP4's role in inflammatory processes?

STEAP4's involvement in inflammatory pathways offers important research opportunities:

• Experimental models:

  • In vitro cytokine stimulation of relevant cell types

  • Animal models of inflammatory conditions

  • Patient-derived samples from inflammatory diseases

• Assessment techniques:

  • Cytokine profiling before and after STEAP4 antibody treatment

  • Immune cell recruitment and activation analysis

  • Signaling pathway interrogation (NF-κB, MAPK, JAK-STAT)

• Functional readouts:

  • Changes in inflammatory gene expression

  • Alterations in immune cell function

  • Tissue-specific inflammatory responses

• Integration with metabolic parameters:

  • STEAP4 integrates inflammatory and metabolic responses

  • Combined analysis of inflammatory markers and metabolic parameters provides comprehensive understanding

  • Assessment of insulin sensitivity and glucose metabolism alongside inflammatory readouts

How can advanced imaging techniques be combined with STEAP4 antibodies for spatial expression analysis?

Spatial analysis of STEAP4 expression offers deeper biological insights:

• Multiplexed immunofluorescence approaches:

  • Sequential staining with different antibodies

  • Spectral unmixing for multiple fluorophores

  • tyramide signal amplification for sensitivity enhancement

• Super-resolution microscopy applications:

  • STORM/PALM for nanoscale localization

  • SIM for improved optical sectioning

  • Label optimization for super-resolution compatibility

• Mass cytometry and imaging mass cytometry:

  • Metal-conjugated STEAP4 antibodies for highly multiplexed analysis

  • Single-cell resolution with dozens of simultaneous markers

  • Spatial context preservation with imaging mass cytometry

• Computational analysis integration:

  • Machine learning for pattern recognition

  • Neighborhood analysis for cellular interactions

  • Correlation with clinical parameters for translational insights

By combining these advanced techniques with STEAP4 antibodies, researchers can gain unprecedented insights into the spatial distribution and functional relationships of STEAP4 in complex biological systems.