SULT2A1 Antibody

What is SULT2A1 and what biological functions does it regulate?

SULT2A1 (Bile salt sulfotransferase) is a cytosolic enzyme that utilizes 3'-phospho-5'-adenylyl sulfate (PAPS) as a sulfonate donor to catalyze the sulfonation of steroids and bile acids primarily in the liver and adrenal glands. It mediates the sulfation of a wide range of substrates including pregnenolone, androsterone, dehydroepiandrosterone (DHEA), bile acids, cholesterol, and various xenobiotics containing alcohol and phenol functional groups . This sulfotransferase plays critical roles in:

• Steroid and lipid homeostasis maintenance

• Bile acid metabolism regulation

• Increasing water solubility of compounds to enhance renal excretion

• Xenobiotic detoxification

• Potential metabolic activation of certain carcinogenic compounds (by similarity)

Alternative names for this enzyme include Dehydroepiandrosterone sulfotransferase (DHEA-ST), Hydroxysteroid Sulfotransferase (HST), ST2, ST2A3, and Sulfotransferase 2A1 (ST2A1) .

What sample types can be analyzed using SULT2A1 antibodies?

SULT2A1 antibodies have demonstrated reactivity with multiple species and sample types:

When selecting samples, researchers should consider:

• Fresh or properly preserved tissues/cells (flash-frozen or fixed according to antibody requirements)

• Inclusion of positive control samples (liver or adrenal tissues)

• Proper extraction protocols to maintain protein integrity

• Species compatibility with the specific antibody clone

How do I optimize Western blot conditions for SULT2A1 detection?

Optimizing Western blot for SULT2A1 detection requires attention to several key parameters:

• Sample preparation: For liver tissue, use RIPA buffer with protease inhibitors. ~20-50 μg total protein typically yields good results.

• Gel percentage and running conditions: 10-12% SDS-PAGE gels are optimal for resolving SULT2A1's ~35 kDa band.

• Transfer conditions:

  • Semi-dry transfer: 15V for 30 minutes

  • Wet transfer: 100V for 60 minutes at 4°C

  • PVDF membranes are recommended over nitrocellulose

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST

  • Anti-SULT2A1 primary antibody dilutions:

    • Polyclonal: 0.5-1 μg/mL (1:500-1:1000)

    • Monoclonal: 1-2 μg/mL (varies by clone)

  • Incubate overnight at 4°C for optimal signal-to-noise ratio

• Detection strategy:

  • HRP-conjugated secondary antibodies with ECL detection

  • Expected band size: ~35 kDa

  • Use reducing conditions and appropriate immunoblot buffer groups

• Controls:

  • Positive control: Human liver lysate

  • Negative control: Tissues with minimal SULT2A1 expression

  • Loading control: β-actin or GAPDH

How can SULT2A1 antibodies be employed in multi-parameter flow cytometry analysis?

Multi-parameter flow cytometry with SULT2A1 antibodies enables sophisticated analysis of expression patterns in heterogeneous cell populations:

• Sample preparation protocol:

  • Single-cell suspensions must be fixed with 4% paraformaldehyde (10 min, room temperature)

  • Permeabilize with 0.1% Triton X-100 or commercial permeabilization buffers

  • Wash cells 3x with PBS containing 1% BSA

• Antibody panel design considerations:

  • SULT2A1 antibodies are available with different conjugates (e.g., Alexa Fluor 555)

  • Pair with surface markers for liver cell populations (e.g., CD45, CD68, EpCAM)

  • Include viability dyes and isotype controls

• Sequential staining approach:

  • Surface marker staining (pre-permeabilization)

  • Fixation and permeabilization

  • Intracellular SULT2A1 staining (typically 1:50-1:200 dilution)

  • Secondary antibody (if using unconjugated primary)

• Data analysis strategies:

  • Gate on viable single cells

  • Identify SULT2A1-positive populations

  • Quantify mean fluorescence intensity

  • Correlate with other markers to identify specific cell subpopulations

This approach is particularly valuable for studying SULT2A1 expression in primary hepatocytes, liver progenitor cells, and during hepatic differentiation processes .

What are the considerations for using SULT2A1 antibodies in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) with SULT2A1 antibodies can reveal protein-protein interactions critical to understanding SULT2A1's biological functions:

• Lysis buffer selection:

  • Gentle lysis buffers (e.g., 1% NP-40, 150mM NaCl, 50mM Tris pH 7.4)

  • Include protease/phosphatase inhibitors and 1mM DTT

  • Avoid harsh detergents that disrupt protein-protein interactions

• Antibody selection criteria:

  • Choose antibodies raised against native epitopes, not denatured proteins

  • Ensure antibodies recognize endogenous protein conformations

  • Polyclonal antibodies often perform better than monoclonals for Co-IP

• Pre-clearing strategy:

  • Pre-clear lysates with protein A/G beads (1-2 hours at 4°C)

  • Remove non-specific binding proteins before adding SULT2A1 antibody

• Controls and validation:

  • IgG control: Use species-matched non-specific IgG

  • Input control: Save 5-10% of pre-IP lysate

  • Reverse Co-IP: Immunoprecipitate with antibodies against suspected interaction partners

  • Validate with recombinant proteins if available

• Detection methods:

  • Western blot using antibodies against suspected interaction partners

  • Mass spectrometry for unbiased identification of binding partners

This technique has revealed interactions between SULT2A1 and various nuclear receptors that regulate its expression and activity in steroid metabolism pathways.

How can genetic variants of SULT2A1 impact antibody-based detection methods?

Genetic variants of SULT2A1 can influence antibody-based detection in important ways:

• SNP-related epitope modifications:

  • The rs2637125 SNP, located near the SULT2A1 coding region, has been associated with serum DHEAS concentrations in genome-wide association studies

  • While functional studies have shown this variant does not affect DHEA/DHEAS ratios, other variants may impact protein structure

• Antibody epitope mapping considerations:

  • Antibodies targeting regions containing polymorphic sites may show differential binding

  • Monoclonal antibodies are more susceptible to epitope changes than polyclonals

  • Western blotting under reducing conditions may mask these differences

• Experimental design recommendations:

  • Include positive controls from characterized cell lines (e.g., HepG2)

  • When studying populations with genetic diversity, validate antibody performance

  • Consider using multiple antibodies targeting different epitopes

  • Sequence the SULT2A1 gene in experimental samples when inconsistent results occur

• Validation approaches:

  • siRNA knockdown to confirm specificity

  • Recombinant protein expression of variant forms

  • Correlation with mRNA expression by RT-PCR

Studies comparing AA, AG, and GG genotypes of rs2637125 found no significant differences in DHEAS, DHEA, androstenedione, cortisol, or cortisone concentrations, suggesting this particular variant does not substantially alter protein function or detection .

How should I design immunohistochemistry experiments for SULT2A1 localization studies?

Immunohistochemistry (IHC) for SULT2A1 localization requires careful consideration of tissue preparation, antibody selection, and detection methods:

• Fixation optimization:

  • Formalin-fixed paraffin-embedded (FFPE) tissues: 10% neutral buffered formalin, 24-48 hours

  • Fresh frozen sections: Fix in cold acetone (10 min) or 4% paraformaldehyde (10-15 min)

  • Antigen retrieval: Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0), 20 minutes at 95-100°C

• Antibody selection and validation:

  • Validate antibody specificity on known positive tissue (liver, adrenal)

  • Optimal dilution determination: Typically 1:100-1:500 for commercial antibodies

  • Include negative controls (no primary antibody, isotype control)

• Detection system optimization:

  • Polymer-based detection systems offer improved sensitivity

  • Chromogenic vs. fluorescent detection based on research needs

  • For multiplexing, use spectrally distinct fluorophores (e.g., Alexa Fluor 555)

• Counterstaining and mounting:

  • Hematoxylin for nuclear counterstain in chromogenic IHC

  • DAPI for nuclear counterstain in fluorescent IHC

  • Use antifade mounting medium for fluorescence preservation

• Analysis and interpretation:

  • Expected pattern: Primarily cytoplasmic localization

  • Quantification methods: H-score, percent positive cells, staining intensity

  • Digital image analysis for objective quantification

SULT2A1 typically shows cytoplasmic staining in hepatocytes and adrenocortical cells, with potential nuclear localization in some contexts that should be validated with subcellular fractionation or co-localization studies .

What controls should be included when validating a new lot of SULT2A1 antibody?

Proper validation of new SULT2A1 antibody lots requires comprehensive controls:

• Positive tissue/cell controls:

  • Human liver tissue (primary expression site)

  • HepG2 human hepatocellular carcinoma cells (known expression)

  • Adrenal tissue (secondary expression site)

  • Mouse kidney tissue (validated reactivity)

• Negative controls:

  • Technical: Omission of primary antibody

  • Biological: Tissues known to lack SULT2A1 expression

  • siRNA/shRNA knockdown samples

  • SULT2A1 knockout samples (if available)

• Specificity tests:

  • Western blot analysis confirming correct molecular weight (~35 kDa)

  • Peptide competition assay using immunizing peptide

  • Cross-reactivity assessment with related sulfotransferases

• Lot-to-lot comparison:

  • Side-by-side testing with previous validated lot

  • Titration series to determine optimal working dilution

  • Signal-to-noise ratio assessment

  • Quantitative comparison of staining intensity

• Application-specific validation:

  • For Western blotting: Confirm band size and intensity

  • For IHC/ICC: Evaluate subcellular localization pattern

  • For flow cytometry: Compare mean fluorescence intensity

  • For IP: Verify pull-down efficiency

Documentation of antibody performance across these validation steps should be maintained as reference for future experiments and troubleshooting.

How can I resolve non-specific binding when using SULT2A1 antibodies in immunofluorescence?

Non-specific binding in immunofluorescence with SULT2A1 antibodies can be addressed through systematic optimization:

• Blocking optimization:

  • Extend blocking time to 1-2 hours at room temperature

  • Test alternative blocking agents: 5-10% normal serum (species of secondary antibody), 3-5% BSA, commercial blockers

  • Add 0.1-0.3% Triton X-100 to blocking buffer for better penetration

• Antibody dilution and incubation:

  • Further dilute primary antibody (start with 1:500, then 1:1000)

  • Extend primary antibody incubation to overnight at 4°C

  • Increase wash steps (5-6 times, 5 minutes each)

  • Dilute secondary antibody further (1:1000-1:2000)

• Fluorophore selection:

  • Choose fluorophores with spectral properties distinct from autofluorescence

  • For liver tissue, avoid green fluorophores (significant autofluorescence)

  • Consider Alexa Fluor 555 or 647 conjugates for better signal-to-noise ratio

• Fixation and permeabilization adjustments:

  • Compare 4% PFA vs. methanol fixation

  • Optimize permeabilization time (5-15 minutes)

  • Consider gentler detergents (e.g., 0.01-0.05% saponin)

• Microscopy settings:

  • Adjust exposure times to minimize background

  • Use spectral unmixing if available

  • Capture negative control images with identical settings

When staining HepG2 cells, researchers have successfully used Anti-SULT2A1 antibodies at 10 μg/mL with a 3-hour room temperature incubation, followed by NorthernLights 557-conjugated secondary antibodies and DAPI counterstaining to achieve specific cytoplasmic localization .

What methodological approaches can address conflicting SULT2A1 expression data between antibody-based and mRNA-based detection?

Discrepancies between antibody-based protein detection and mRNA-based SULT2A1 expression require systematic investigation:

• Validation of antibody specificity:

  • Western blot confirmation of single band at expected molecular weight

  • Knockdown/knockout validation

  • Comparison across multiple antibodies targeting different epitopes

• Technical considerations for protein detection:

  • Protein extraction efficiency (membranous proteins require specialized buffers)

  • Post-translational modifications affecting epitope recognition

  • Protein stability and half-life differences

• mRNA measurement validation:

  • Primer design and specificity (check for alternative splice variants)

  • Reference gene selection for normalization

  • RNA quality assessment (RIN score >7 recommended)

• Biological explanations for discrepancies:

  • Post-transcriptional regulation (miRNAs, RNA-binding proteins)

  • Translational efficiency differences

  • Protein stability and degradation rates

  • Cell-specific or subcellular localization differences

• Integrative approaches:

  • Single-cell analysis to detect cellular heterogeneity

  • Pulse-chase experiments to assess protein turnover

  • Polysome profiling to examine translational efficiency

  • Proteasome inhibition to assess degradation contributions

When investigating SULT2A1 genetic variants, researchers found no correlation between genotype and DHEA/DHEAS ratios despite association with serum DHEAS in GWAS studies, highlighting the importance of integrating multiple methodological approaches .

How do post-translational modifications affect SULT2A1 detection with antibodies?

Post-translational modifications (PTMs) of SULT2A1 can significantly impact antibody recognition and experimental outcomes:

• Common PTMs affecting SULT2A1:

  • Phosphorylation (activating or inhibitory)

  • Acetylation

  • Ubiquitination (regulating protein stability)

  • Glycosylation (affecting solubility and localization)

• Epitope-specific considerations:

  • Antibodies raised against synthetic peptides may miss PTM-dependent epitopes

  • Modifications near the epitope can sterically hinder antibody binding

  • Some PTMs may create neo-epitopes recognized by specific antibodies

• Experimental strategies:

  • Phosphatase treatment before Western blotting to remove phosphorylation

  • Use of PTM-specific antibodies (e.g., phospho-SULT2A1)

  • 2D gel electrophoresis to separate PTM variants

  • Mass spectrometry to map and quantify PTMs

• Sample preparation recommendations:

  • Include phosphatase inhibitors when studying phosphorylation

  • Use fresh samples when possible (PTMs can be lost during storage)

  • Consider native conditions for maintaining PTM integrity

• Validation approaches:

  • In vitro modification with specific enzymes

  • Site-directed mutagenesis of PTM sites

  • Correlation with functional activity assays

Understanding the PTM status of SULT2A1 is particularly important when studying its role in drug metabolism and steroid homeostasis, as modifications can alter substrate specificity and catalytic efficiency.

How can SULT2A1 antibodies be used to investigate liver disease progression and drug-induced liver injury?

SULT2A1 antibodies offer valuable tools for investigating hepatic pathologies:

• Expression pattern analysis in disease states:

  • Decreased SULT2A1 expression occurs in certain liver diseases

  • Zonal distribution changes may precede overt pathology

  • Multiplexing with markers of liver injury (e.g., α-SMA, collagen)

• Methodological approaches:

  • Tissue microarray analysis of large sample cohorts

  • Sequential liver biopsies to track expression changes

  • Co-localization with cell-type specific markers

  • Quantitative image analysis for precise expression measurement

• Drug-induced liver injury applications:

  • SULT2A1 changes may predict drug metabolism alterations

  • Models for investigating sulfonation-dependent drug toxicity

  • Correlation with clinical chemistry markers (ALT, AST, bilirubin)

• Experimental design considerations:

  • Include appropriate disease controls

  • Stage-specific sampling to track progression

  • Correlation with functional metabolic assessments

  • Integration with genomic/transcriptomic data

• Translational research opportunities:

  • Biomarker development for early detection

  • Personalized medicine applications based on SULT2A1 status

  • Therapeutic targeting of sulfotransferase pathways

SULT2A1 immunostaining in HepG2 human hepatocellular carcinoma cells shows specific cytoplasmic localization that can be altered under disease conditions or xenobiotic exposure, providing a valuable model system for investigating regulation in liver disease contexts .

What are the methodological considerations for studying SULT2A1 genetic variants in population-based research?

Population studies of SULT2A1 genetic variants require careful methodological planning:

• Sample selection and cohort design:

  • Population stratification considerations

  • Sample size calculations based on variant frequency

  • Longitudinal vs. cross-sectional approaches

  • Family-based studies for inheritance patterns

• Genotyping approaches:

  • SNP selection (functional vs. tagging SNPs)

  • Consider rs2637125 and rs182420 variants

  • Whole-gene sequencing for rare variant detection

  • Validation with multiple genotyping methods

• Phenotyping considerations:

  • Standardized hormone measurements (LC-MS/MS recommended)

  • DHEA/DHEAS ratio as sulfonation capacity marker

  • Additional relevant steroids (cortisol, androstenedione)

  • Time of sampling (circadian variation)

• Statistical analysis frameworks:

  • Quantile regression models for comparing median hormone levels

  • Sex-stratified analyses (hormonal differences)

  • Multiple testing correction

  • Haplotype analysis for linked variants

• Validation strategies:

  • Replication in independent cohorts

  • Functional studies of identified variants

  • In vitro enzyme activity assessments

  • Integration with transcriptomic data

In a population-based study with 3,300 participants, researchers identified 43 individuals homozygous for the minor allele of SNP rs2637125 (AA) and compared them with matched AG and GG genotype carriers. Despite GWAS associations with DHEAS levels, no significant differences were found in DHEA/DHEAS ratios or individual hormone concentrations across genotypes, highlighting the importance of functional validation of genetic associations .

How will emerging antibody technologies advance SULT2A1 research in the coming years?

Emerging antibody technologies are poised to transform SULT2A1 research in several key ways:

• Single-cell analysis applications:

  • Mass cytometry (CyTOF) with metal-conjugated anti-SULT2A1 antibodies

  • Spatial transcriptomics combined with antibody detection

  • Single-cell Western blotting for heterogeneity assessment

  • In situ sequencing with antibody detection

• Advanced imaging technologies:

  • Super-resolution microscopy for subcellular localization

  • Intravital imaging with near-infrared fluorophore conjugates

  • CLARITY and tissue clearing methods for 3D visualization

  • Live-cell imaging with non-perturbing antibody fragments

• Functional antibody applications:

  • Conformation-specific antibodies detecting active enzyme states

  • Intrabodies for tracking intracellular dynamics

  • Nanobodies for improved tissue penetration

  • Bispecific antibodies for co-localization studies

• High-throughput screening approaches:

  • Antibody arrays for pathway activation profiling

  • Automated imaging platforms for drug screening

  • Microfluidic antibody delivery systems

  • Organ-on-chip models with integrated immunodetection

• Therapeutic and diagnostic potential:

  • Antibody-drug conjugates targeting SULT2A1-expressing tumors

  • Companion diagnostics for drugs metabolized by SULT2A1

  • Non-invasive imaging with radiolabeled antibodies

  • Immunomodulation of SULT2A1 pathways