HSD3B7 Antibody

What is HSD3B7 and what is its biological function in research models?

HSD3B7 (3 beta-hydroxysteroid dehydrogenase type 7) belongs to the 3-beta-HSD enzyme family and plays a crucial role in bile acid biosynthesis. It catalyzes the conversion of 3β-hydroxy-Δ5 bile acid intermediates to 3α-hydroxy-Δ4 bile acids and is predominantly expressed in the liver .

The enzyme is known by several alternative names:

• 3 beta-hydroxysteroid dehydrogenase type VII

• 3-beta-HSD VII

• 3-beta-hydroxy-Delta(5)-C27 steroid oxidoreductase

• C(27) 3-beta-HSD

• Cholest-5-ene-3-beta,7-alpha-diol 3-beta-dehydrogenase

In knockout mouse models, HSD3B7 deficiency affects the bile acid synthesis pathway with complex compensatory mechanisms . Recent research has also identified HSD3B7 as a potential biomarker in clear cell renal cell carcinoma (ccRCC), with elevated expression correlating with poor prognosis and aggressive tumor characteristics .

What applications are validated for HSD3B7 antibodies and what are the experimental considerations?

HSD3B7 antibodies have been validated for multiple research applications across different sample types:

Application notes:

• For IHC, TE buffer pH 9.0 is recommended for antigen retrieval, though citrate buffer pH 6.0 can be used as an alternative

• For Western blot, the observed molecular weight is typically 33-36 kDa, which differs from the calculated weight of 41 kDa

• Polyclonal antibodies from rabbit hosts are most commonly available and validated

What are the species reactivity profiles available for HSD3B7 antibodies?

Different HSD3B7 antibodies offer varying species reactivity profiles:

When selecting an antibody for cross-species applications, researchers should verify the epitope conservation across species and critically evaluate validation data provided by manufacturers . Some commercially available antibodies target specific amino acid regions that may be more or less conserved across species. For example, antibodies targeting amino acids 131-197 have been validated for human, mouse, and rat reactivity .

How should HSD3B7 antibodies be handled and stored for optimal performance?

Improper storage and handling of HSD3B7 antibodies can significantly impact their performance. Follow these research-validated guidelines:

Recommended storage conditions:

• Store at -20°C for long-term preservation (up to 12 months)

• For frequent use over 1 month, store at 4°C to minimize freeze-thaw cycles

• Store in aliquots to avoid repeated freeze-thaw cycles which can degrade antibody performance

Buffer composition:

• Typically stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some formulations may contain 0.5% BSA as a stabilizer

Handling precautions:

• Avoid vortexing antibody solutions; mix by gentle inversion or pipetting

• Centrifuge briefly before opening vials to collect solution at the bottom

• Allow solutions to equilibrate to room temperature before opening to prevent condensation

• Document lot numbers and maintain controlled freeze-thaw records for experimental reproducibility

Antibody performance should be validated after extended storage periods, particularly for critical applications like quantitative analysis in clinical research.

What purification methods are used for commercial HSD3B7 antibodies and how do they affect applications?

Commercial HSD3B7 antibodies undergo different purification methods that influence their specificity, background, and optimal application range:

Affinity purification methods:

• Immunogen/antigen affinity chromatography is the most common method

• Protein A/G purification may be used for IgG antibodies

Impact of purification on applications:

• Antigen-affinity purified antibodies:

  • Higher specificity for the target epitope

  • Reduced background in immunostaining applications

  • Optimal for IHC and IF where specificity is critical

  • Examples include antibodies purified against recombinant HSD3B7 protein

• Protein A/G purified antibodies:

  • May contain a broader range of antibodies recognizing different epitopes

  • Sometimes preferred for immunoprecipitation applications

  • May provide stronger signals in Western blotting

Purity considerations:

• Antibody purity is typically ≥95% as determined by SDS-PAGE

• Higher purity antibodies generally provide more reproducible results across experiments

• For specialized applications (e.g., structural studies), higher purity antibodies may be required

When selecting an HSD3B7 antibody, researchers should consider how the purification method aligns with their specific application needs and evaluate validation data for their intended application.

How do I optimize HSD3B7 antibody protocols for detecting low expression levels in tissue samples?

Detecting low levels of HSD3B7 expression requires systematic optimization beyond standard protocols:

Antigen retrieval optimization:

• Test multiple buffers with detailed pH gradients:

  • TE buffer pH 9.0 (primary recommendation)

  • Citrate buffer pH 6.0 (alternative approach)

  • Tris-EDTA with 0.05% Tween-20

• Optimize retrieval time and temperature using a matrix approach:

  • Temperature range: 95-125°C

  • Time range: 10-30 minutes

  • Document optimization results systematically

Signal amplification strategies:

• Implement tyramide signal amplification (TSA):

  • Provides 10-50× signal enhancement

  • Critical protocol modifications:

    • Reduce primary antibody concentration (1:200-1:1000)

    • Include hydrogen peroxide quenching step

    • Optimize TSA reagent concentration and incubation time

• For Western blotting:

  • Use PVDF membranes with 0.2μm pore size (vs. standard 0.45μm)

  • Implement enhanced chemiluminescence with femto-sensitive substrates

  • Consider antibody-HRP direct conjugates to eliminate secondary antibody variables

Advanced controls for low-expression validation:

• Parallel analysis of HSD3B7 mRNA by in situ hybridization or RT-qPCR

• Concentration-matched IgG controls for each tissue type

• Quantitative standard curve using recombinant HSD3B7 protein (for Western blot)

• Comparison across multiple antibodies targeting different HSD3B7 epitopes

Digital image acquisition optimization:

• For fluorescence applications:

  • Use spectral unmixing to separate autofluorescence

  • Implement deconvolution algorithms

  • Extend exposure times with multiple frame averaging

• For chromogenic IHC:

  • Use multispectral imaging

  • Implement digital enhancement algorithms with standardized controls

These optimizations must be systematically documented and validated with appropriate positive and negative controls to ensure reproducibility and reliability of low-level HSD3B7 detection.

How can I validate the specificity of HSD3B7 antibodies in knockout or knockdown models?

Rigorous validation of HSD3B7 antibodies using genetic depletion models is essential for definitive specificity confirmation:

Knockout model validation strategy:
HSD3B7 knockout mice provide the gold standard for antibody validation. As demonstrated in published research, these models show complete replacement of the HSD3B7 gene, resulting in undetectable protein expression .

• Tissue preparation matrix:

  • Compare wild-type, heterozygous, and homozygous knockout liver samples

  • Process samples using identical protocols

  • Include additional tissues with variable expression

• Multi-assay validation approach:

  • Western blot: Expected pattern shows strong band at ~33 kDa in wild-type, ~50% reduction in heterozygous, and complete absence in knockout samples

  • IHC: Positive hepatocyte staining in wild-type liver with complete absence in knockout tissue

  • Immunofluorescence: Similar pattern with subcellular resolution

• Secondary antibody and non-specific binding controls:

  • Include secondary-only controls for each genotype

  • Implement peptide competition assays using immunizing peptide

  • Document background staining patterns systematically

siRNA knockdown validation for human samples:
When knockout models aren't available (especially for human samples), siRNA knockdown provides a valuable alternative :

• Knockdown optimization:

  • Test multiple siRNA sequences targeting different regions of HSD3B7 mRNA

  • Verify knockdown efficiency at mRNA level by RT-qPCR

  • Establish time-course of protein depletion (typically 48-96h)

• Quantitative analysis requirements:

  • Measure band intensity reduction in Western blot (typically 70-90% reduction with effective siRNA)

  • Implement statistical analysis across multiple independent experiments

  • Use scrambled siRNA controls matched for GC content

• Functional validation:

  • Correlate phenotypic changes with HSD3B7 depletion

  • Rescue experiments with siRNA-resistant HSD3B7 expression constructs

Document critical validation parameters:

• Antibody lot number, dilution, and incubation conditions

• Complete protein extraction and separation protocols

• Image acquisition settings with unprocessed original images

• Quantification methodology with statistical analysis

This comprehensive validation approach ensures that signals detected by HSD3B7 antibodies represent the intended target protein rather than cross-reactive species or background artifacts.

What are the latest research findings on HSD3B7's role in cancer progression and how can antibodies contribute to this research?

Recent integrated analysis of single-cell and bulk RNA sequencing data has revealed critical roles for HSD3B7 in cancer biology, particularly in clear cell renal cell carcinoma (ccRCC) :

HSD3B7 as a prognostic biomarker:

Functional role in cancer progression:
Experimental studies using siRNA-mediated knockdown of HSD3B7 in ccRCC cell lines demonstrated :

• Cell proliferation: Significant reduction in HSD3B7-depleted cells

• Cell cycle: G1 phase arrest following HSD3B7 knockdown

• Apoptosis: Increased percentage of apoptotic cells

• Migration and invasion: Reduced capabilities in transwell and wound healing assays

Antibody applications in cancer research:

• Diagnostic and prognostic applications:

  • IHC-based tissue microarray analysis for patient stratification

  • Correlation with clinical outcomes in retrospective studies

  • Multi-marker panels including HSD3B7

• Mechanistic investigations:

  • Subcellular localization changes in tumor progression

  • Protein-protein interaction studies in cancer cells

  • Post-translational modification analysis

• Therapeutic development applications:

  • Target validation in preclinical models

  • Antibody-drug conjugate development

  • Response biomarker for emerging therapies

Research methodology considerations:

• Use multiple antibodies targeting different epitopes to confirm findings

• Include careful controls for specificity in tumor tissues

• Correlate protein expression with transcriptomic data

• Consider context-dependent functions in different cancer types

This emerging research suggests HSD3B7 may be both a valuable prognostic biomarker and potential therapeutic target in ccRCC, with antibody-based detection methods playing a central role in advancing this field.

How do I troubleshoot cross-reactivity issues with HSD3B7 antibodies?

Cross-reactivity represents a significant challenge in HSD3B7 antibody applications due to sequence homology with other 3-beta-HSD family members. A systematic troubleshooting approach is essential:

Identify potential cross-reactive proteins:

• Sequence homology analysis:

  • Align HSD3B7 sequence with other 3-beta-HSD family members (HSD3B1, HSD3B2, etc.)

  • Focus on the specific epitope region targeted by your antibody

  • Example: Antibodies targeting amino acids 121-170 may have different cross-reactivity profiles than those targeting 281-369

• Expression pattern mapping:

  • Document tissue expression profiles of all 3-beta-HSD family members

  • Identify tissues expressing only HSD3B7 vs. tissues expressing multiple family members

  • Use these tissues for systematic validation

Experimental validation of specificity:

• Multi-technique approach:

  Technique	Methodology	Expected Result for Specific Antibody
  Western blot	Run samples with known expression of HSD3B family members	Single band at ~33-36 kDa for HSD3B7
  Peptide competition	Pre-incubate antibody with excess immunizing peptide	Complete signal abolishment
  Knockout/knockdown controls	Test on HSD3B7-depleted samples	Absence of signal
  Immunodepletion	Sequential immunoprecipitation to remove HSD3B7	No remaining reactivity

• Antibody comparison:

  • Test multiple antibodies targeting different HSD3B7 epitopes

  • Compare monoclonal vs. polyclonal antibodies

  • Evaluate antibodies from different host species

Optimization strategies for reducing cross-reactivity:

• Buffer and condition modifications:

  • Increase washing stringency with higher salt concentration (150mM → 250-300mM NaCl)

  • Add low concentrations of detergents (0.1-0.3% Triton X-100)

  • Modify blocking solutions (try protein-free blockers)

• Dilution optimization:

  • Perform careful titration series (e.g., 1:100, 1:500, 1:1000, 1:2000)

  • Balance signal strength vs. specificity

  • Document optimal dilution for each application systematically

• Alternative detection strategies:

  • Consider highly-specific detection methods (e.g., Proximity Ligation Assay)

  • Use orthogonal approaches (mass spectrometry) for validation

  • Implement multi-color imaging to distinguish specific from non-specific signals

Documentation and reporting:

• Keep detailed records of all troubleshooting experiments

• Document antibody lot numbers and exact experimental conditions

• Report cross-reactivity issues to antibody manufacturers

• Include comprehensive specificity controls in publications

This systematic approach allows researchers to identify and mitigate cross-reactivity issues, ensuring reliable and reproducible results with HSD3B7 antibodies.

What are the recommended protocols for using HSD3B7 antibodies in co-immunoprecipitation studies of protein interaction networks?

Co-immunoprecipitation (Co-IP) is valuable for investigating HSD3B7 protein interactions, but requires careful optimization for this membrane-associated protein:

Sample preparation optimization:

• Tissue selection:

  • Liver tissue offers highest endogenous expression

  • Cell lines with verified HSD3B7 expression (e.g., 769-P cells)

• Membrane protein solubilization strategy:

  • Pre-clear cellular debris at low speed (1,000g) before main clarification

  • Maintain samples at 4°C throughout processing

  • Consider mild sonication (3-5 short pulses) to enhance extraction

Immunoprecipitation protocol:

• Input standardization:

  • Use 1-3 mg total protein per IP reaction

  • Quantify protein concentration using BCA or Bradford assay

  • Reserve 5% input sample before antibody addition

• Antibody binding:

  • Use 0.5-4.0 μg HSD3B7 antibody per reaction

  • Incubate overnight at 4°C with gentle rotation

  • Pre-couple antibody to beads for potentially improved results

• Bead selection and handling:

  • Protein A/G magnetic beads offer advantages over agarose for membrane proteins

  • Use 30-50 μL bead slurry per reaction

  • Block beads with BSA before use to reduce non-specific binding

• Washing optimization:

  • Perform 4-5 washes with rotation for 5 minutes each

  • Final wash with detergent-free buffer

Essential controls:

• Negative controls:

  • Species-matched non-immune IgG

  • "Beads-only" control to identify non-specific binding proteins

  • Pre-immune serum if available

• Specificity controls:

  • Peptide competition (pre-incubate antibody with immunizing peptide)

  • HSD3B7 knockdown or knockout samples

  • Reverse IP with antibodies against suspected interaction partners

Detection and analysis:

• Western blot detection:

  • Use 10-12% gels for optimal HSD3B7 separation

  • Transfer to PVDF membranes (preferable for hydrophobic proteins)

  • Block with 5% BSA rather than milk for membrane proteins

  • Primary antibody: Use different HSD3B7 antibody (different epitope) at 1:500-1:1000

  • Consider enhanced chemiluminescence detection for maximum sensitivity

• Mass spectrometry analysis:

  • On-bead digestion may provide better results than elution for membrane proteins

  • Include IgG controls for background subtraction

  • Use label-free quantification to identify enriched proteins

  • Validate top hits by reverse Co-IP and functional studies

This optimized protocol addresses the specific challenges of working with HSD3B7 as a membrane-associated protein while providing robust controls to ensure specificity and reproducibility of interaction data.