sult1st3 Antibody

What is SULT1A3 and what role does it play in human physiology?

SULT1A3 belongs to the cytosolic sulfotransferase (SULT) family of Phase II drug-metabolizing enzymes. These enzymes catalyze the transfer of a sulfonate group from 3′-phosphoadenosine 5′-phosphosulfate to various endogenous and xenobiotic compounds. SULT1A3 specifically sulfates catecholamine neurotransmitters, distinguishing it from other SULT family members with different substrate specificities. The enzyme plays an important role in neurotransmitter metabolism and in the biotransformation of numerous compounds in the human body . SULT1A3 is also known by several alternative names including HAST, HAST3, M-PST, ST1A3, ST1A3/ST1A4, ST1A5, STM, and TL-PST, reflecting its discovery history and functional characteristics .

What is the tissue distribution pattern of SULT1A3 in human brain?

SULT1A3 shows a distinct expression pattern across different regions of the human brain. Immunoblot analyses reveal that SULT1A3 expression is highest in the superior temporal gyrus, hippocampus, and temporal lobe regions. Interestingly, there appears to be an inverse relationship between SULT1A3 and SULT1A1 expression in many brain regions - areas with high SULT1A3 expression often show lower SULT1A1 expression and vice versa. The frontal lobe is somewhat unique in showing high expression of both isoforms . This specific distribution pattern likely reflects the functional roles of SULT1A3 in neurotransmitter metabolism in different brain regions.

How is SULT1A3 distributed at the cellular level in brain tissue?

SULT1A3 is expressed in both neurons and glial cells in the human brain, but its expression is not uniform across all cells. Immunohistochemical analyses show that only select neurons and glial cells are immunoreactive for SULT1A3. The differential immunoreactivity may be associated with factors such as epitope availability, substrate distribution, neuronal subtype, and synaptic status . Notably, studies with cultured human SH-SY5Y neurons and CCF-STTG1 astrocytes did not detect SULT1A3 protein expression, suggesting that expression may be limited to specific neural cell subtypes rather than occurring constitutively in all brain cells .

What considerations should be made when selecting an antibody for SULT1A3 detection?

When selecting a SULT1A3 antibody, researchers should consider several critical factors. First, evaluate the specificity of the antibody – SULT1A1 and SULT1A3 share 93% sequence identity, making cross-reactivity a significant concern . Antibodies raised against full-length SULT1A3 protein may recognize multiple SULT1A isoforms, while peptide antibodies designed against unique sequences (such as the 83-97 amino acid region) can provide greater specificity . Second, consider the application requirements - some antibodies perform better in western blots while others are optimized for immunohistochemistry or immunofluorescence. Third, examine validation data showing the antibody's performance in your specific application and tissue type. Finally, consider the antibody format (monoclonal vs polyclonal) and host species to ensure compatibility with your experimental design, particularly for co-staining experiments.

How can I validate the specificity of a SULT1A3 antibody?

Validating SULT1A3 antibody specificity requires a multi-step approach:

• Positive and negative controls: Test the antibody against recombinant SULT1A3 protein and tissue/cells known to express or lack SULT1A3.

• Cross-reactivity testing: Compare reactivity against related SULT family members, particularly SULT1A1, given their high sequence similarity. In published work, anti-SULT1A3 peptide antibody showed only slight reactivity to SULT1A1, while exhibiting some cross-reactivity with SULT1C2 .

• Knockdown/knockout validation: Test antibody on samples where SULT1A3 expression has been reduced via siRNA or CRISPR technologies.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples - specific staining should be eliminated.

• Multiple antibody comparison: Use different antibodies targeting distinct SULT1A3 epitopes to confirm staining patterns.

• Band migration analysis: In western blots, verify that the detected band migrates at the expected molecular weight (~34 kDa for SULT1A3).

For meaningful validation, researchers should normalize data to account for differences in antibody affinities when comparing SULT1A expression patterns across different tissues .

What protocols are recommended for immunohistochemical detection of SULT1A3 in brain tissue?

For optimal immunohistochemical detection of SULT1A3 in brain tissue sections, the following protocol is recommended based on published research:

• Sample preparation: Fix tissue appropriately (e.g., 4% paraformaldehyde) and prepare sections (paraffin-embedded or frozen).

• Antigen retrieval: For formalin-fixed tissues, use heat-induced epitope retrieval methods to restore antigenicity.

• Blocking: Block for 1-4 hours or overnight at 4°C using SEA Block or similar product to reduce non-specific binding .

• Primary antibody incubation: Dilute anti-SULT1A3 antibody in PBE (phosphate-buffered saline with 500 mM EDTA, 1% bovine serum albumin, pH 7.6) at a ratio of 1:100 and incubate on slides for 1 hour in humidity chambers .

• Washing: Wash thoroughly in appropriate buffer (e.g., TBST) to remove unbound antibody.

• Secondary antibody: Apply appropriately labeled secondary antibody (e.g., HRP-conjugated).

• Detection: Develop using a suitable chromogen or fluorescent detection system.

• Co-staining: For cell-type identification, include markers such as anti-neuronal nuclei or anti-glial fibrillary acid protein antibodies .

• Controls: Always include negative controls (primary antibody omitted) and positive controls (tissues known to express SULT1A3).

Particular attention should be paid to antibody dilution and incubation times, as these may need optimization for specific antibodies and tissue samples.

What is the recommended western blot protocol for SULT1A3 detection?

Based on published research, the following western blot protocol is recommended for SULT1A3 detection:

• Sample preparation: Prepare cytosolic fractions from tissues of interest using appropriate homogenization and fractionation techniques.

• Protein separation: Separate proteins using SDS-PAGE (10-12% gels typically work well for SULT1A3's ~34 kDa size).

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane.

• Blocking: Block membranes for 4 hours to overnight at 4°C in absolute SEA Block (or similar blocking solution) .

• Primary antibody: After washing three times in TBST, incubate membranes overnight at 4°C with anti-SULT1A3 primary antibody diluted 1:1000 in 0.1% milk in TBST .

• Washing: Wash membranes twice for 5 minutes in TBST at room temperature.

• Secondary antibody: Incubate with anti-rabbit HRP-conjugated secondary antibody diluted 1:50,000 in 0.1% milk in TBST for 1 hour at room temperature .

• Detection: Develop using chemiluminescent substrate (West Pico or Fempto SuperSignal) and expose to autoradiograph film or digital imager .

• Quantification: For relative quantitation, use densitometry software like Un-Scan-IT to analyze band intensity, normalizing to appropriate loading controls .

When comparing SULT1A1 and SULT1A3 levels, consider normalizing data to account for potential differences in antibody affinities.

How can I differentiate between SULT1A3 and other SULT family members in my samples?

Differentiating between SULT1A3 and other SULT family members requires careful experimental design:

• Use selective antibodies: Employ antibodies raised against unique peptide sequences, such as the EVNDPGEPSGLETLK (83-97) region used for generating SULT1A3-specific antibodies .

• Electrophoretic migration patterns: Although SULT family members have similar molecular weights, slight differences in migration can be used for identification. For example, while the SULT1A3 peptide antibody showed cross-reactivity with SULT1C2, these isoforms can be distinguished by their different electrophoretic migration patterns .

• Enzyme activity assays: Supplement immunodetection with enzymatic activity assays using substrates selective for SULT1A3 (e.g., dopamine) versus those preferred by other SULT isoforms.

• Mass spectrometry validation: For definitive identification, consider using mass spectrometry-based proteomic approaches to confirm the identity of immunoreactive bands.

• Recombinant protein standards: Include purified recombinant SULT1A3 and related isoforms as standards on your blots for comparison.

• Sequential immunoprecipitation: Deplete samples of specific SULT isoforms sequentially to identify remaining immunoreactive species.

Remember that SULT1A1 and SULT1A3 share 93% sequence identity, making absolute specificity challenging with many antibodies .

How does SULT1A3 expression correlate with neurodegenerative disease pathology?

This question represents an active area of research, as SULT1A3's role in catecholamine metabolism makes it potentially relevant to neurodegenerative conditions. While the provided search results don't directly address SULT1A3 in neurodegenerative diseases, researchers investigating this question would approach it by:

• Comparative expression analysis: Compare SULT1A3 expression levels in brain regions affected by specific neurodegenerative diseases versus unaffected regions and control samples using validated antibodies.

• Cell-type specific analysis: Determine whether disease-related changes in SULT1A3 expression are global or restricted to specific neuronal or glial populations, using co-staining techniques with cell-type markers and SULT1A3 antibodies.

• Functional correlations: Correlate SULT1A3 expression or activity with levels of catecholamine neurotransmitters, their metabolites, and markers of oxidative stress in affected tissues.

• Genetic association studies: Examine whether polymorphisms in the SULT1A3 gene correlate with disease risk, progression, or treatment response.

• Animal models: Use SULT1A3 antibodies to characterize expression changes in animal models of neurodegenerative diseases, correlating these with behavioral and pathological outcomes.

The regional distribution of SULT1A3 in the human brain, with highest expression in the superior temporal gyrus, hippocampus, and temporal lobe , makes it particularly interesting for studies of conditions affecting these regions, such as Alzheimer's disease and temporal lobe epilepsy.

What are the current methodologies for studying SULT1A3 enzyme activity in conjunction with protein expression?

Advanced research on SULT1A3 often requires correlating protein expression detected by antibodies with enzyme activity. Current methodologies include:

• Radiometric assays: Measure transfer of radiolabeled sulfate from 35S-PAPS (3'-phosphoadenosine 5'-phosphosulfate) to SULT1A3 substrates like dopamine. After separation of substrate from product, quantify radioactivity to determine enzyme activity.

• HPLC-based assays: Detect and quantify sulfated metabolites produced by SULT1A3 using high-performance liquid chromatography, often coupled with mass spectrometry.

• Colorimetric/fluorometric assays: Use p-nitrophenol sulfation or similar approaches where product formation can be measured spectrophotometrically.

• In situ enzyme activity: Develop techniques to visualize SULT1A3 activity in tissue sections using activity-based probes, correlating with antibody staining in adjacent sections.

• Co-immunoprecipitation-activity assays: Immunoprecipitate SULT1A3 using validated antibodies and measure enzyme activity in the precipitated fraction.

• Single-cell correlation: Develop methods to correlate SULT1A3 immunofluorescence intensity with enzyme activity at the single-cell level.

In such studies, researchers should consider the thermolabile nature of SULT1A3 enzyme activity in their experimental design, particularly regarding sample handling temperatures and stability.

What are common pitfalls in SULT1A3 antibody-based experiments and how can they be addressed?

Researchers working with SULT1A3 antibodies should be aware of these common pitfalls and solutions:

• Cross-reactivity issues: Due to high sequence similarity with SULT1A1 (93% identity) , many antibodies cross-react with both proteins. Solution: Use peptide-specific antibodies targeting unique regions, and include appropriate controls with recombinant SULT1A1 and SULT1A3 proteins.

• Inconsistent cellular localization: SULT1A3 may not be uniformly expressed in all neurons or glial cells . Solution: Include appropriate cell-type markers and recognize that negative staining doesn't necessarily indicate technical failure.

• Lack of signal in cultured cells: Some cultured cell lines (e.g., SH-SY5Y neurons and CCF-STTG1 astrocytes) may not express detectable SULT1A3 . Solution: Validate cell models before extensive experimentation, and consider using primary cells or tissue sections.

• Antibody batch variation: Different lots of the same antibody may show performance variations. Solution: Validate each new lot against previous lots and maintain reference samples.

• Epitope masking: Post-translational modifications or protein-protein interactions may mask antibody epitopes. Solution: Try multiple antibodies targeting different regions of SULT1A3 and optimize antigen retrieval methods.

• Background signals: High background may obscure specific signals. Solution: Optimize blocking conditions (e.g., using absolute SEA Block ) and antibody dilutions.

• Quantification challenges: When comparing SULT1A1 and SULT1A3, differences in antibody affinities may confound results. Solution: Normalize data using purified recombinant protein standards .

How should researchers interpret variations in SULT1A3 expression across different brain regions?

The interpretation of SULT1A3 expression variations across brain regions requires careful consideration of several factors:

• Functional significance: Higher SULT1A3 expression in the superior temporal gyrus, hippocampus, and temporal lobe suggests potentially greater need for catecholamine metabolism in these regions. Researchers should correlate expression patterns with known regional differences in neurotransmitter levels and turnover.

• Inverse relationship with SULT1A1: The observed inverse pattern between SULT1A1 and SULT1A3 expression in many brain regions suggests complementary functions. When interpreting high or low SULT1A3 expression, consider concurrent SULT1A1 levels, as one may compensate for the other.

• Cell-type distribution: Variations may reflect differences in the proportions of SULT1A3-expressing cell types rather than global changes in expression. Always complement regional analyses with cell-type specific investigations.

• Substrate availability: Interpret SULT1A3 expression in the context of regional substrate availability. High expression may correlate with regions rich in dopamine or other catecholamine neurotransmitters.

• Technical considerations: Ensure that apparent regional differences aren't due to technical factors like tissue fixation variability, antibody penetration, or section thickness. Always include internal controls.

• Individual variation: Consider inter-individual variations when interpreting regional differences. The provided research shows variations "between brain sections and among brains" .

• Disease context: In disease-related studies, interpret regional changes in the context of known pathological processes affecting those regions.

What quantitative methods best represent SULT1A3 expression changes in comparative studies?

For accurate quantitative analysis of SULT1A3 expression, researchers should consider these methodological approaches:

• Relative quantitation with densitometry: For western blots, use densitometry software (e.g., Un-Scan-IT) to quantify band intensity, normalizing to appropriate housekeeping proteins. When comparing SULT1A1 and SULT1A3, normalize data to account for potential differences in antibody affinities .

• Absolute quantification with recombinant standards: Include purified recombinant SULT1A3 protein at known concentrations to generate standard curves for more accurate quantification.

• Image analysis for immunohistochemistry: Use digital image analysis with appropriate software to quantify immunostaining intensity, area, and cell counts in tissue sections. Ensure consistent acquisition parameters across all samples.

• qPCR correlation: Complement protein detection with quantitative PCR to measure SULT1A3 mRNA levels, allowing correlation between transcriptional and translational regulation.

• Single-cell analysis: For heterogeneous tissues like brain, consider single-cell approaches (flow cytometry, single-cell Western blotting, or imaging mass cytometry) to quantify SULT1A3 in specific cell populations.

• Multiplex detection systems: Employ multiplex detection platforms to simultaneously quantify SULT1A3 along with related proteins or cell-type markers.

• Statistical analysis: Apply appropriate statistical methods for comparative studies, considering biological and technical replicates, and adjusting for multiple comparisons when necessary.

• Normalization strategies: In addition to protein loading controls, consider normalizing to total cell number, tissue weight, or other relevant parameters depending on the experimental context.

Table 1: Comparative SULT1A3 and SULT1A1 Expression and Function in Human Brain