HSD11B2 Antibody

What is HSD11B2 and what biological role does it play?

HSD11B2 (hydroxysteroid 11-beta dehydrogenase 2) is a microsomal enzyme complex that catalyzes the conversion of biologically active cortisol to inactive cortisone. This enzyme plays a critical role in regulating intracellular glucocorticoid levels, thereby protecting the nonselective mineralocorticoid receptor from occupation by glucocorticoids . The enzyme is encoded by the HSD11B2 gene (Gene ID: 3291) and has a calculated molecular weight of approximately 44 kDa (405 amino acids), though the observed molecular weight in experimental conditions typically ranges from 40-43 kDa . HSD11B2 is primarily localized in the cytoplasm and is expressed in tissues such as kidney and placenta, where it plays crucial roles in hormone regulation .

What types of HSD11B2 antibodies are currently available for research applications?

Current research-grade HSD11B2 antibodies are predominantly rabbit polyclonal antibodies, though they vary in their specific epitope targets and applications. Common variants include:

• Antibodies targeting the middle region (AA 284-314)

• Antibodies targeting amino acids 151-250

• Antibodies developed against full-length protein (AA 1-405)

• N-terminal and C-terminal specific antibodies

Most available antibodies are unconjugated and purified through protein A columns, often followed by peptide affinity purification . These antibodies typically display reactivity against human samples, with many also cross-reacting with mouse and rat HSD11B2. Some demonstrate broader cross-reactivity with species such as horse, cow, guinea pig, rabbit, and sheep samples .

What is the difference between using monoclonal versus polyclonal antibodies for HSD11B2 detection?

While the search results primarily describe polyclonal antibodies, it's important to understand the methodological differences between antibody types. Polyclonal antibodies like those described in the search results (14192-1-AP, bs-3618R, CAB8077) recognize multiple epitopes on the HSD11B2 protein, providing higher sensitivity but potentially lower specificity .

For HSD11B2 research, polyclonal antibodies offer advantages when:

• Detecting low-abundance protein in tissues like kidney and placenta

• Studying species variants due to potential cross-reactivity

• Performing protein detection under denaturing conditions (Western blot)

The trade-off is that batch-to-batch variation can occur with polyclonal antibodies. Research requiring absolute epitope specificity might benefit from monoclonal antibodies, though these would need validation for specific applications. When selecting an antibody for HSD11B2 research, consider the experimental conditions, detection method, and specific research question to determine whether the broader epitope recognition of polyclonal antibodies outweighs the consistency of monoclonals for your particular application.

What are the optimal dilutions and conditions for using HSD11B2 antibodies in different applications?

The optimal dilution of HSD11B2 antibodies varies by application and specific antibody. Based on the search results, here is a comprehensive table of recommended dilutions:

For optimal results, it's recommended to titrate these antibodies in each testing system as results can be sample-dependent . For IHC applications with antibody 14192-1-AP, antigen retrieval with TE buffer pH 9.0 is suggested, with citrate buffer pH 6.0 as an alternative option .

What positive controls should be used when validating HSD11B2 antibodies?

When validating HSD11B2 antibodies, the following positive controls have been confirmed effective:

• Tissue samples: Human placenta tissue, human kidney tissue, and mouse kidney tissue have been validated as positive controls for Western blot applications

• Cell lines: Transfected HEK-293 cells expressing HSD11B2 serve as excellent positive controls

• Knockdown/Knockout validation: Several publications have utilized knockdown/knockout systems for validating antibody specificity, as noted in the search results

When selecting a positive control, consider the species reactivity of your antibody. For example, antibody 14192-1-AP has demonstrated reactivity with human, mouse, and rat samples, while bs-3618R shows reactivity with mouse, rat, and horse samples . The expected molecular weight for HSD11B2 is 40-43 kDa, which serves as an important verification parameter for Western blot applications .

What sample preparation methods are recommended when working with HSD11B2 antibodies?

Effective sample preparation is critical for obtaining reliable results with HSD11B2 antibodies. For Western blot applications, the following methodological considerations should be implemented:

• Tissue homogenization: For kidney or placental tissue, use a buffer containing protease inhibitors to prevent degradation of HSD11B2 protein, which is sensitive to proteolytic cleavage

• Protein extraction: Since HSD11B2 is primarily localized in the cytoplasm , cytoplasmic extraction protocols are suitable

• Sample denaturation: Standard denaturation with SDS and reducing agents is compatible with the antibodies described in the search results

• Loading control: Include appropriate loading controls specific to the subcellular fraction being analyzed

For immunohistochemistry, the search results indicate:

• Fixation: Paraformaldehyde fixation is compatible with these antibodies

• Antigen retrieval: For antibody 14192-1-AP, use TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative

• Blocking: Standard blocking with serum or BSA is suitable for reducing background

The storage conditions for these antibodies are typically -20°C, and they remain stable for one year after shipment. Aliquoting is unnecessary for -20°C storage of antibody 14192-1-AP . For long-term storage, avoid repeated freeze-thaw cycles to maintain antibody performance .

How can I troubleshoot weak or absent HSD11B2 signal in Western blots?

When troubleshooting weak or absent HSD11B2 signal in Western blots, consider the following methodological approaches:

• Optimization of antibody concentration: While the recommended dilution ranges are provided (1:1000-1:4000 for 14192-1-AP; 1:300-5000 for bs-3618R), initially use a higher concentration (less diluted) and then optimize for specific samples

• Sample preparation adjustments:

  • Ensure adequate protein extraction from tissues known to express HSD11B2 (kidney, placenta)

  • Include protease inhibitors to prevent degradation

  • Try different lysis buffers if the standard protocol yields inconsistent results

• Detection system evaluation:

  • Enhance sensitivity by using high-sensitivity chemiluminescent substrates

  • Consider longer exposure times for weak signals

  • Try signal amplification systems for low-abundance samples

• Technical considerations:

  • Verify transfer efficiency using reversible staining methods

  • Ensure proper primary antibody binding time (overnight at 4°C may improve signal)

  • Check antibody storage conditions and expiration dates

• Positive controls: Include known positive controls such as human kidney or placenta tissue or transfected HEK-293 cells expressing HSD11B2

If signal remains weak despite these adjustments, consider that HSD11B2 expression levels vary significantly between tissues and may be influenced by experimental conditions or disease states being studied.

What are the common artifacts or non-specific bindings observed with HSD11B2 antibodies and how can they be minimized?

Non-specific binding can complicate interpretation of results when using HSD11B2 antibodies. Common artifacts and methodological solutions include:

• Multiple bands in Western blots:

  • The expected molecular weight of HSD11B2 is 40-43 kDa

  • Bands at other molecular weights may represent non-specific binding, proteolytic fragments, or isoforms

  • Increase blocking time or blocking agent concentration (e.g., 5% BSA instead of 3%)

  • Perform additional washes with increased Tween-20 concentration (0.1% to 0.3%)

• Background staining in immunohistochemistry:

  • Optimize antigen retrieval conditions (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Increase blocking time and concentration

  • Dilute primary antibody further (within recommended ranges: 1:20-1:200 for 14192-1-AP, 1:200-400 for bs-3618R)

  • Use species-specific blocking reagents to minimize cross-reactivity

• Validation through controls:

  • Include negative controls (omitting primary antibody)

  • Use knockdown/knockout samples as specificity controls

  • Pre-absorb antibody with immunizing peptide if available

• Cross-reactivity considerations:

  • Be aware of potential cross-reactivity with related proteins, especially when using polyclonal antibodies

  • Verify results with a second antibody targeting a different epitope of HSD11B2

These methodological adjustments can significantly improve signal-to-noise ratio and increase confidence in the specificity of observed signals.

How do I interpret differences in HSD11B2 staining patterns between tissue types?

Interpreting differences in HSD11B2 staining patterns requires understanding its biological distribution and regulatory mechanisms. Key methodological considerations include:

• Tissue-specific expression levels:

  • HSD11B2 is predominantly expressed in kidney and placenta tissues

  • Expression levels may vary significantly between tissues, requiring adjusted exposure times or detection sensitivity

  • Different tissues may require optimization of antibody dilutions within the recommended ranges

• Subcellular localization:

  • HSD11B2 is primarily localized in the cytoplasm

  • Variations in subcellular distribution between tissues may reflect tissue-specific functions

  • Use appropriate subcellular markers in co-localization studies to confirm localization patterns

• Physiological context interpretation:

  • HSD11B2 expression can be regulated by hormonal status and pathological conditions

  • Compare experimental samples with appropriate controls from similar physiological states

  • Document experimental conditions that might affect expression (time of day, stress levels, medication status)

• Analytical approaches:

  • Quantify relative expression between tissues using appropriate image analysis software

  • Normalize to cell number or tissue area when comparing different samples

  • Use statistical methods appropriate for the distribution of your data

When reporting differences between tissues, include comprehensive methodological details about sample collection, processing, and analysis parameters to ensure reproducibility.

How can HSD11B2 antibodies be utilized in studies of cortisol metabolism disorders?

HSD11B2 antibodies serve as valuable tools for investigating cortisol metabolism disorders, particularly those involving dysregulation of the cortisol-cortisone conversion. Methodological approaches include:

• Expression analysis in disease models:

  • Western blot quantification of HSD11B2 protein levels in tissues from patients or animal models with suspected enzyme dysfunction

  • Immunohistochemical analysis to identify altered cellular distribution in pathological states

  • Flow cytometric analysis of intracellular HSD11B2 using permeabilization protocols and antibodies suitable for FACS

• Correlation with clinical parameters:

  • Compare HSD11B2 expression levels with cortisol/cortisone ratios in blood or urine

  • Correlate tissue expression patterns with disease severity metrics

  • Use standardized quantification methods to enable comparisons across studies

• Mechanistic investigations:

  • Combine antibody detection with functional assays measuring enzymatic activity

  • Use in experimental interventions (pharmacological or genetic) targeting the enzyme

  • Apply in co-immunoprecipitation experiments to identify interacting proteins that may regulate function

• Translational applications:

  • Evaluate HSD11B2 as a biomarker for conditions such as apparent mineralocorticoid excess or hypertension

  • Assess changes in expression in response to therapeutic interventions

  • Screen for compounds that modulate HSD11B2 expression or activity

These approaches provide comprehensive analysis of HSD11B2's role in disease pathogenesis and potential therapeutic interventions targeting cortisol metabolism.

What protocols are recommended for co-localization studies involving HSD11B2 and other proteins?

For co-localization studies involving HSD11B2 and other proteins, the following methodological protocols are recommended:

• Immunofluorescence optimization:

  • Use antibodies suitable for immunofluorescence applications (multiple antibodies in the search results are validated for IF)

  • For antibody bs-3618R, use dilutions of 1:50-200 for IF applications

  • Consider sequential staining protocols if antibodies are raised in the same host species

• Confocal microscopy protocol:

  • Use multi-channel acquisition to simultaneously detect HSD11B2 and target proteins

  • Employ appropriate controls for channel bleed-through

  • Collect z-stack images to analyze three-dimensional co-localization

  • Use standardized co-localization coefficients (Pearson's, Mander's) for quantitative analysis

• Proximity ligation assay (PLA) methodology:

  • For detecting proteins in close proximity (<40 nm)

  • Combine HSD11B2 antibodies with antibodies against potential interacting proteins

  • Quantify interaction signals using appropriate image analysis software

• Sample preparation considerations:

  • For tissue sections, use thin sections (5-7 μm) to minimize background

  • For cultured cells, optimize fixation methods to preserve antigenicity while maintaining cellular structure

  • Consider optical clearing techniques for thick tissue sections

• Controls and validation:

  • Include single-stained controls for each antibody

  • Use known interacting proteins as positive controls

  • Consider super-resolution microscopy techniques for validation of close associations

These protocols enable precise analysis of HSD11B2's spatial relationship with other proteins, providing insights into functional interactions and regulatory mechanisms.

How can HSD11B2 antibodies be applied in drug development research targeting glucocorticoid regulation?

HSD11B2 antibodies offer valuable applications in drug development research targeting glucocorticoid regulation through several methodological approaches:

• Target engagement studies:

  • Assess changes in HSD11B2 expression levels following drug treatment using Western blot analysis with antibodies such as 14192-1-AP (1:1000-1:4000 dilution)

  • Evaluate alterations in subcellular localization using immunofluorescence with antibodies validated for IF applications

  • Quantify expression changes in high-throughput screening using ELISA-based methods

• Mechanism of action investigations:

  • Use immunoprecipitation to identify drug-induced changes in protein-protein interactions

  • Combine with activity assays to correlate expression levels with functional changes

  • Assess post-translational modifications that might be affected by drug candidates

• Biomarker development:

  • Evaluate HSD11B2 expression as a potential pharmacodynamic biomarker

  • Develop and validate immunoassays for clinical sample testing

  • Correlate tissue expression with circulating metabolite ratios

• Predictive toxicology applications:

  • Screen compounds for unintended effects on HSD11B2 expression

  • Assess tissue-specific modulation using immunohistochemistry protocols (1:20-1:200 dilution for 14192-1-AP)

  • Identify potential mechanisms of adverse effects related to glucocorticoid dysregulation

• Translational research strategies:

  • Validate preclinical findings in human samples

  • Develop companion diagnostic approaches for targeted therapies

  • Establish standardized protocols for assessment across different research phases

These methodological approaches support comprehensive evaluation of compounds targeting the HSD11B2 pathway, enabling efficient drug development and potential identification of novel therapeutic strategies for conditions involving glucocorticoid dysregulation .

What are the key differences in detecting HSD11B2 in different species samples?

Detection of HSD11B2 across different species requires consideration of sequence homology, antibody cross-reactivity, and species-specific expression patterns. Methodological considerations include:

When working with different species:

• Antibody selection strategy:

  • Choose antibodies raised against conserved epitopes for cross-species applications

  • Verify sequence homology between the immunogen and target species

  • Consider antibodies like 14192-1-AP that have demonstrated multi-species reactivity

• Validation methodology:

  • Always validate antibodies in your species of interest before proceeding to experimental samples

  • Include appropriate positive controls from the same species

  • Consider optimization of sample preparation methods for species-specific tissues

• Detection system adjustments:

  • Species-specific secondary antibodies should be employed

  • Optimize blocking conditions to minimize background in different species samples

  • Consider higher primary antibody concentrations for species with lower sequence homology

These methodological approaches will maximize the likelihood of successful HSD11B2 detection across diverse experimental models.

How should I approach epitope mapping when studying HSD11B2 protein interactions?

Epitope mapping for HSD11B2 protein interaction studies requires strategic selection of antibodies targeting different regions of the protein. Based on the search results, several antibodies targeting different epitopes are available:

• Available epitope-specific antibodies:

  • Middle region (AA 284-314): ABIN950176

  • AA 151-250 region: bs-3618R

  • N-terminal and C-terminal specific antibodies

  • Full-length protein (AA 1-405)

• Methodological approach for epitope mapping:

  • Use multiple antibodies targeting different regions to determine accessible epitopes in native protein

  • Compare antibody binding under different conditions (native vs. denatured)

  • Employ competitive binding assays to identify overlapping epitopes

  • Consider peptide arrays for fine mapping of specific binding sites

• Protein interaction studies:

  • Select antibodies targeting non-interacting regions to avoid interfering with binding partners

  • Use epitope-tagged HSD11B2 constructs alongside antibody detection for validation

  • Consider proximity-based assays (FRET, PLA) with strategically selected antibody pairs

  • Perform pull-down assays with antibodies targeting different epitopes and compare interactome profiles

• Structural considerations:

  • Correlate epitope accessibility with predicted protein structure

  • Consider post-translational modifications that might affect antibody binding

  • Evaluate epitope conservation across species for evolutionary studies

This comprehensive approach enables detailed characterization of HSD11B2 structural domains involved in protein interactions and provides insights into functional regulation mechanisms.

What methodological considerations are important when using HSD11B2 antibodies in high-throughput screening applications?

Implementing HSD11B2 antibodies in high-throughput screening (HTS) applications requires optimization for reliability, reproducibility, and efficiency. Key methodological considerations include:

• Assay format selection:

  • ELISA-based detection: Use antibodies validated for ELISA applications (bs-3618R at 1:500-1000 dilution)

  • Cell-based immunofluorescence: Consider antibodies validated for IF/ICC (bs-3618R at 1:50-200)

  • Flow cytometry: ABIN950176 has been validated for FACS applications

• Optimization for automation:

  • Standardize sample preparation protocols

  • Establish reproducible antibody dilution methods

  • Determine optimal incubation times that balance signal development with throughput

  • Implement quality control metrics for batch-to-batch consistency

• Signal detection and quantification:

  • Optimize signal-to-noise ratio through systematic testing of blocking conditions

  • Establish detection thresholds based on positive and negative controls

  • Implement appropriate normalization methods for plate-to-plate comparisons

  • Consider automated image analysis for cell-based assays

• Validation requirements:

  • Include on-plate controls for antibody performance

  • Validate hits with orthogonal methods

  • Implement Z-factor calculations to assess assay quality

  • Consider multiplexing with additional markers to increase information content

• Data management and analysis:

  • Develop standardized data capture and analysis pipelines

  • Implement appropriate statistical methods for hit identification

  • Consider machine learning approaches for pattern recognition in complex datasets

  • Establish clear criteria for follow-up validation

These methodological considerations ensure robust implementation of HSD11B2 antibodies in high-throughput screening applications, facilitating efficient discovery in areas such as drug development, pathway analysis, and biomarker identification.

How are HSD11B2 antibodies being utilized in studies of cortisol-related pathologies beyond traditional applications?

Recent advances in HSD11B2 research have expanded its relevance beyond classical mineralocorticoid regulation. Emerging methodological applications include:

• Neuroscience applications:

  • Detection of HSD11B2 in brain regions involved in stress response and neuroinflammation

  • Investigation of blood-brain barrier integrity through HSD11B2 expression in cerebrovascular endothelium

  • Correlation of neural HSD11B2 expression with behavioral phenotypes in stress models

• Metabolic disorder research:

  • Analysis of adipose tissue expression patterns in obesity and metabolic syndrome

  • Investigation of liver-specific regulation in non-alcoholic fatty liver disease

  • Correlation of skeletal muscle expression with insulin resistance parameters

• Cancer biology:

  • Evaluation as a potential biomarker in hormone-responsive tumors

  • Investigation of expression changes during cancer progression and metastasis

  • Assessment of HSD11B2 in tumor microenvironment regulation

• Developmental biology:

  • Temporal expression patterns during organogenesis

  • Role in glucocorticoid programming of developmental trajectories

  • Epigenetic regulation of HSD11B2 in developmental contexts

For these emerging applications, researchers should consider antibodies with validated tissue-specific performance and appropriate controls. Combining protein detection with functional assays provides the most comprehensive understanding of HSD11B2's role in these diverse pathological contexts.

What are the current challenges and potential solutions in developing therapeutic antibodies targeting HSD11B2?

While the search results focus on research antibodies, exploration of therapeutic antibodies targeting HSD11B2 presents several methodological challenges and potential solutions:

• Target accessibility challenges:

  • HSD11B2 is primarily intracellular (cytoplasmic) , making it inaccessible to conventional therapeutic antibodies

  • Potential solutions include antibody-drug conjugates, cell-penetrating antibody technologies, or targeting extracellular domains if present

• Specificity requirements:

  • High homology with HSD11B1 necessitates exquisite specificity

  • Solution approaches include extensive screening against related dehydrogenases

  • Structure-based design targeting unique epitopes (e.g., middle region AA 284-314)

• Functional modulation strategies:

  • Developing antibodies that modulate enzyme activity rather than just binding

  • Screening for conformation-specific antibodies that lock the enzyme in active/inactive states

  • Considering allosteric modulation through antibody binding to regulatory domains

• Delivery methodologies:

  • Explore tissue-specific targeting strategies for kidney or other HSD11B2-expressing tissues

  • Consider antibody formats optimized for tissue penetration (e.g., single-domain antibodies)

  • Investigate alternative delivery systems for intracellular targeting

• Validation frameworks:

  • Develop appropriate in vitro and in vivo models for efficacy testing

  • Establish clinically relevant endpoints related to cortisol/cortisone ratios

  • Design biomarker strategies to monitor target engagement

These methodological approaches could guide development of novel therapeutic strategies targeting HSD11B2 in conditions characterized by dysregulated cortisol metabolism.

What are the recommended storage and handling protocols to ensure antibody stability and performance?

Proper storage and handling of HSD11B2 antibodies is critical for maintaining their stability and ensuring reproducible experimental results. Based on the search results, the following methodological recommendations apply:

• Storage conditions:

  • Store at -20°C for long-term stability

  • Antibody 14192-1-AP remains stable for one year after shipment when stored properly

  • Antibody bs-3618R should be stored at -20°C to maintain stability

• Aliquoting recommendations:

  • For antibody 14192-1-AP, aliquoting is unnecessary for -20°C storage

  • For other antibodies, consider aliquoting to avoid repeated freeze-thaw cycles

  • Use sterile tubes and aseptic technique when preparing aliquots

• Handling protocols:

  • Avoid repeated freeze-thaw cycles

  • When removing from storage, thaw antibodies on ice

  • Centrifuge briefly before opening to collect solution at the bottom of the tube

  • Return to storage promptly after use

• Dilution and working solution stability:

  • Prepare working dilutions immediately before use

  • For antibody 14192-1-AP, the storage buffer is PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • For antibody bs-3618R, the storage buffer is 0.01M TBS (pH 7.4) with 1% BSA, 0.02% Proclin300, and 50% Glycerol

• Quality control measures:

  • Include positive controls in each experiment to verify antibody performance

  • Document lot numbers and maintain records of antibody performance

  • Consider periodic validation of stored antibodies against fresh antibody preparations

These storage and handling protocols will help ensure consistent antibody performance and experimental reproducibility across studies.

How should researchers validate new lots of HSD11B2 antibodies before incorporating them into established protocols?

Rigorous validation of new antibody lots is essential for maintaining experimental consistency and data reliability. A comprehensive methodological approach to HSD11B2 antibody validation includes:

• Side-by-side comparison protocol:

  • Run parallel experiments with previous and new antibody lots

  • Use identical samples, concentrations, and experimental conditions

  • Quantitatively compare signal intensity, background levels, and specificity

• Performance metric evaluation:

  • For Western blot: Compare band intensity at the expected molecular weight (40-43 kDa) , signal-to-noise ratio, and detection of any non-specific bands

  • For IHC/IF: Assess staining intensity, pattern specificity, and background levels

  • For ELISA/FACS: Compare standard curves, dynamic range, and detection limits

• Validation using established controls:

  • Positive controls: Human placenta tissue, human kidney tissue, mouse kidney tissue, or transfected HEK-293 cells expressing HSD11B2

  • Negative controls: Tissues or cells known not to express HSD11B2

  • Specificity controls: Knockdown/knockout samples if available

• Titration experiments:

  • Determine optimal working concentrations for each application

  • Compare titration curves between old and new lots

  • Establish new working dilutions if necessary, within recommended ranges (e.g., 1:1000-1:4000 for WB with 14192-1-AP)

• Documentation practices:

  • Record detailed validation results including images and quantitative measurements

  • Document lot numbers, receipt dates, and expiration dates

  • Maintain validation records for reference and troubleshooting

This systematic validation approach ensures experimental continuity when transitioning between antibody lots and provides documentation for quality control purposes.

What methods can be used to quantify and standardize HSD11B2 expression levels across different experimental systems?

Standardization of HSD11B2 quantification across diverse experimental systems requires rigorous methodological approaches:

• Absolute quantification methods:

  • Implement quantitative Western blotting using purified recombinant HSD11B2 protein standards

  • Develop and validate ELISA methods with defined standard curves

  • Consider mass spectrometry-based approaches for absolute protein quantification

• Relative quantification strategies:

  • Normalize to appropriate housekeeping proteins (consistent across experimental systems)

  • Use digital image analysis with standardized acquisition parameters

  • Implement densitometry with background subtraction and linear range verification

• Internal calibration approaches:

  • Include common reference samples across experiments

  • Use calibrator samples with known HSD11B2 content

  • Implement bridging studies when comparing across different experimental platforms

• Standardized reporting practices:

  • Report both raw and normalized data

  • Document complete methodological details including antibody information, dilutions, and detection systems

  • Include validation metrics such as coefficients of variation

  • Specify dynamic range of detection methods

• Cross-platform validation:

  • Verify findings using orthogonal methods (e.g., protein vs. mRNA expression)

  • Compare antibody-based methods with activity assays when applicable

  • Use multiple antibodies targeting different epitopes for confirmation