SULT1C2 Antibody

What is SULT1C2 and what significance does it have in biological research?

SULT1C2 is a sulfotransferase enzyme that catalyzes the sulfation of various endogenous and xenobiotic compounds. Recent research has revealed several critical biological functions of SULT1C2:

• Modification of xenobiotic compounds to enhance secretion, though this process can sometimes render these compounds carcinogenic in contexts such as tobacco exposure

• Conversion of cholesterol to cholesterol sulfate in mitochondrial membranes, thereby increasing mitochondrial respiratory capacity and membrane potential

• Involvement in cancer progression, particularly in lung adenocarcinoma (LUAD) related to tobacco exposure and hepatocellular carcinoma (HCC)

SULT1C2 expression correlates significantly with lung adenocarcinoma patient survival in smokers and appears to be epigenetically regulated through DNA methylation patterns that may vary across different ethnic populations . In HCC, SULT1C2 has been shown to promote cell growth, migration, and invasiveness .

What are the standard applications for SULT1C2 antibodies in research?

SULT1C2 antibodies are employed in multiple research techniques:

• Western blotting/immunoblotting to detect and quantify SULT1C2 protein levels, using antibodies such as SC-130274

• Immunohistochemistry to visualize SULT1C2 expression patterns in tissue sections

• Immunofluorescence for subcellular localization studies, particularly when investigating mitochondrial localization

• Immunoprecipitation for purifying SULT1C2 and studying protein-protein interactions

• ChIP (Chromatin Immunoprecipitation) assays for studying transcription factors that regulate SULT1C2 expression, such as the Aryl Hydrocarbon Receptor (AHR)

For mitochondrial studies, SULT1C2 antibodies have been particularly valuable in confirming the presence of this enzyme in mitochondrial fractions, which has led to discoveries about its role in mitochondrial respiration and membrane potential .

What is the recommended protocol for SULT1C2 detection in Western blotting?

For optimal SULT1C2 detection in Western blotting:

• Sample preparation:

  • For cellular studies: Lyse cells in RIPA buffer with protease inhibitors

  • For mitochondrial studies: Isolate mitochondria using differential centrifugation and verify fraction purity

• Protein separation:

  • Load 20-40 μg protein per lane on 10-12% SDS-PAGE gels

  • Include molecular weight markers covering the 25-45 kDa range (SULT1C2 is approximately 35-36 kDa)

• Transfer and blocking:

  • Transfer to PVDF or nitrocellulose membranes (100V for 1 hour or 30V overnight at 4°C)

  • Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Antibody incubation:

  • Incubate with primary SULT1C2 antibody (1:500 to 1:2000 dilution) overnight at 4°C

  • Wash thoroughly with TBST (3-5 times, 5 minutes each)

  • Incubate with appropriate HRP-conjugated secondary antibody (1:5000 to 1:10000) for 1 hour

• Detection and analysis:

  • Develop using ECL substrate and detect via chemiluminescence imaging

  • Always include appropriate loading controls (e.g., actin as used in SULT1C2 studies)

  • For quantification, perform densitometric analysis of band intensity normalized to loading controls

How should I approach the validation of SULT1C2 antibody specificity?

Comprehensive SULT1C2 antibody validation should include:

• Genetic approaches:

  • siRNA/shRNA knockdown: Verify signal reduction after SULT1C2 knockdown, as demonstrated in HCC studies

  • Overexpression: Confirm increased signal in cells transfected with SULT1C2 expression plasmids

  • CRISPR/Cas9 knockout: Generate true negative controls where possible

• Biochemical validation:

  • Pre-absorption tests: Pre-incubate antibody with purified SULT1C2 protein

  • Peptide competition assays: Demonstrate that the immunizing peptide blocks antibody binding

  • Western blot: Confirm a single band of the expected molecular weight (35-36 kDa)

• Cross-reactivity assessment:

  • Test against related SULT family members, particularly SULT1C2A and SULT1B1 which have been detected in overlapping tissues

  • Proteomic analysis can help identify the specificity of antibodies in complex samples, as demonstrated in mitochondrial studies where SULT1C2 and SULT1C2A peptides were identified

• Multi-technique validation:

  • Confirm that signals from different detection methods (Western blot, IHC, IF) show consistent patterns

  • Correlate protein detection with mRNA expression where possible

How can I use SULT1C2 antibodies to investigate its role in xenobiotic metabolism?

For studying SULT1C2's role in xenobiotic metabolism:

What approaches are recommended for studying SULT1C2's role in mitochondrial function?

To investigate SULT1C2's mitochondrial functions:

• Mitochondrial localization and purification:

  • Isolate highly purified mitochondria using differential centrifugation

  • Confirm SULT1C2 presence in mitochondrial fractions via Western blot and proteomic analysis

  • Use immunofluorescence microscopy with mitochondrial markers to visualize co-localization

• Functional respiratory studies:

  • Measure oxygen consumption rate (OCR) in isolated mitochondria with and without added SULT1C2 and PAPS (3-phosphoadenosine 5-phosphosulfate, the sulfate donor)

  • Assess response to respiratory substrates, ADP, and inhibitors

  • Compare effects of recombinant SULT1C2 to direct addition of cholesterol sulfate

• Membrane studies:

  • Extract mitochondrial lipids and analyze by thin-layer chromatography

  • Quantify cholesterol and cholesterol sulfate levels using lipidomics

  • The relative migration (Rf) values help identify cholesterol sulfate (Rf ≈ 0.52) versus cholesterol (Rf ≈ 0.95-0.97)

• In vivo approaches:

  • Deliver SULT1C2 expression plasmids to target tissues (as demonstrated in kidney studies)

  • Isolate mitochondria from transduced tissues to measure changes in membrane potential and respiratory capacity

  • Assess protection against stress conditions like ischemia/reperfusion injury

Table 1 shows the relative migration (Rf) values for cholesterol and cholesterol sulfate, which is critical for identification in thin-layer chromatography analysis:

How does SULT1C2 expression differ across tissues and what implications does this have for antibody selection?

SULT1C2 exhibits tissue-specific expression patterns with important implications for antibody selection:

• Tissue-specific expression profiles:

  • Lung: Expression levels correlate with tobacco exposure, with higher expression in smokers compared to non-smokers

  • Liver: Elevated in hepatocellular carcinoma compared to normal liver tissue

  • Kidney: Present in mitochondrial fractions, with increased levels after ischemic preconditioning

  • Normal adult lung: Generally low expression with the promoter typically methylated

• Antibody selection considerations:

  • Sensitivity requirements: For tissues with low baseline expression (like normal lung), highly sensitive detection methods and antibodies are essential

  • Specificity: In tissues where multiple SULT isoforms are expressed (e.g., kidney), antibodies must be validated for minimal cross-reactivity with other SULT family members

  • Application optimization: For each tissue type, antibody concentration and detection methods may need specific optimization

• Subcellular localization differences:

  • Mitochondrial localization in kidney tissue requires special consideration for isolation protocols and antibody access to epitopes

  • Nuclear vs. cytoplasmic distribution may vary by tissue type and cellular state

What methods are recommended for quantifying SULT1C2 levels in different experimental conditions?

For precise quantification of SULT1C2:

• Western blot quantification:

  • Use digital imaging systems with linear detection range

  • Normalize SULT1C2 band intensity to appropriate loading controls

  • Include standard curves using recombinant SULT1C2 for absolute quantification

  • Analyze multiple biological replicates for statistical validity

• Comparative expression analysis:

  • For xenobiotic exposure studies, perform dose-response experiments with multiple concentrations of compounds like CSC

  • For cancer studies, compare expression between tumor and adjacent non-tumor tissues

  • For mitochondrial studies, quantify the ratio of cholesterol sulfate to cholesterol as shown in Table 2

Table 2: Effect of SULT1C2 on Cholesterol Sulfate Levels in Isolated Mitochondria

• Mass spectrometry-based quantification:

  • Use targeted MS approaches for absolute quantification

  • Focus on unique peptides that distinguish SULT1C2 from other family members

  • Employ isotope-labeled peptide standards for precise quantification

How can I investigate the epigenetic regulation of SULT1C2 expression using antibodies?

For studying epigenetic regulation of SULT1C2:

• DNA methylation analysis:

  • The SULT1C2 promoter has been shown to be hypomethylated in certain populations (e.g., Asian patients) compared to others (e.g., Caucasians)

  • Correlate methylation patterns with SULT1C2 expression levels

  • In vitro methylation of the SULT1C2 promoter has been shown to significantly decrease transcriptional activity of reporter plasmids

• Chromatin regulation studies:

  • Use ChIP assays with antibodies against histone modifications associated with active (H3K4me3, H3K27ac) or repressed (H3K27me3, H3K9me3) chromatin

  • Target the SULT1C2 promoter region, particularly the area containing the AHR binding site that spans critical methylation sites

  • Compare histone modification patterns between tissues with high and low SULT1C2 expression

• Transcription factor binding analysis:

  • CSC exposure has been shown to increase AHR binding to predicted binding sites in the SULT1C2 promoter

  • Investigate how DNA methylation affects transcription factor binding

  • Use antibodies against AHR and other relevant transcription factors in ChIP assays

• Expression modulation studies:

  • Treat cells with epigenetic modifiers (e.g., 5-Aza-2′-deoxycytidine)

  • Monitor changes in SULT1C2 expression via Western blot and qRT-PCR

  • Research has shown that SULT1C2 expression can be activated by such demethylating agents in cell lines where the promoter is hypermethylated

What techniques can be employed to study SULT1C2's role in cancer progression?

For investigating SULT1C2 in cancer:

How can I develop co-immunoprecipitation protocols to study SULT1C2 interactions with mitochondrial proteins?

For mitochondrial SULT1C2 co-IP studies:

• Mitochondrial isolation and preparation:

  • Isolate highly purified mitochondria using differential centrifugation

  • Verify fraction purity using markers for different compartments

  • Use gentle lysis conditions to preserve protein-protein interactions (e.g., 0.5% CHAPS instead of stronger detergents)

• Antibody selection and validation:

  • Test multiple SULT1C2 antibodies for IP efficiency in mitochondrial samples

  • Validate antibody specificity using Western blot and proteomic analysis

  • Consider using antibodies against both SULT1C2 and potential interaction partners for reciprocal co-IP

• Experimental controls:

  • Include input control (pre-IP mitochondrial lysate)

  • Use IgG control (same species and concentration as IP antibody)

  • Include negative controls such as mitochondria from tissues not expressing SULT1C2

• Detection and validation:

  • Western blot analysis for targeted detection of specific interaction partners

  • Mass spectrometry for unbiased discovery of novel interactions

  • Validate key interactions with multiple methods and reciprocal co-IP

  • Investigate whether interactions are affected by the presence of substrates like cholesterol or PAPS

• Functional validation:

  • Test whether identified interactions affect SULT1C2's ability to convert cholesterol to cholesterol sulfate

  • Investigate how these interactions influence mitochondrial respiration and membrane potential

  • Determine if interactions are affected by stress conditions such as ischemia/reperfusion

What methods can be used to investigate the enzymatic activity of SULT1C2 in different subcellular compartments?

For analyzing SULT1C2 enzymatic activity across subcellular compartments:

• Subcellular fractionation:

  • Separate cellular components into cytosolic, nuclear, mitochondrial, and microsomal fractions

  • Verify fraction purity using established markers

  • Quantify SULT1C2 distribution across fractions by Western blot

• Activity assays in isolated compartments:

  • For mitochondrial activity: Measure conversion of cholesterol to cholesterol sulfate using thin-layer chromatography and lipidomics

  • The identified Rf values for cholesterol sulfate (0.48-0.52) versus cholesterol (0.94-0.97) provide reliable markers for activity assessment

  • Validate activity by comparing native mitochondrial fractions to those supplemented with recombinant SULT1C2 and PAPS

• Functional consequences assessment:

  • For mitochondria: Measure oxygen consumption rate before and after SULT1C2 activity

  • Research has shown that addition of SULT1C2 and PAPS results in increased maximal respiratory capacity in response to succinate, ADP, and rotenone

  • Compare effects of enzymatic activity to direct addition of end products (e.g., cholesterol sulfate)

• In vivo validation:

  • Use gene delivery of SULT1C2 expression plasmids to target tissues

  • Isolate subcellular fractions to measure compartment-specific activities

  • Research in kidney models has shown that SULT1C2 gene delivery increases mitochondrial membrane potential and confers resistance to ischemia/reperfusion injury

How should I approach cross-species analysis of SULT1C2 using antibodies?

For cross-species SULT1C2 research:

• Antibody selection considerations:

  • Perform sequence alignment between human SULT1C2 and target species homologs

  • Focus on the epitope region recognized by the antibody (request this information from vendors)

  • SULT1C2 antibodies have been successfully used in rat models for mitochondrial studies

  • Consider generating species-specific antibodies for critical experiments

• Validation approaches:

  • Western blot validation using tissue samples from the target species

  • Include positive control tissues known to express SULT1C2 in the target species

  • Proteomic analysis can confirm antibody specificity in non-human samples

  • In rat kidney studies, proteomic analysis identified SULT1C2 and SULT1C2A as the predominant SULT isoforms in mitochondrial fractions

• Functional conservation testing:

  • Compare enzymatic activities across species

  • In rat kidney mitochondria, SULT1C2 has been shown to produce cholesterol sulfate similar to predicted human activity

  • Test whether functional roles (e.g., in mitochondrial respiration) are conserved across species

• Data interpretation cautions:

  • Subcellular localization may differ between species

  • Expression patterns and regulation mechanisms may vary significantly across species

  • Be aware that other SULT family members (like SULT1B1) may have different expression profiles across species

How can I troubleshoot non-specific binding issues with SULT1C2 antibodies?

For resolving non-specific binding:

• Antibody dilution optimization:

  • Perform titration experiments to determine optimal antibody concentration

  • For Western blots, test dilutions from 1:500 to 1:2000

  • For immunohistochemistry, test dilutions from 1:50 to 1:200

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat milk, normal serum)

  • Increase blocking time (from 1 hour to overnight)

  • For mitochondrial studies, specifically optimize blocking conditions to reduce background in this enriched fraction

• Washing optimization:

  • Increase washing time and/or number of washes

  • Add mild detergents to wash buffers

  • Use high-salt buffers for more stringent washing

• Sample preparation improvements:

  • For Western blots, ensure complete denaturation of proteins

  • For mitochondrial preparations, confirm high purity of fractions

  • Consider alternative fixation methods for immunohistochemistry

• Control experiments:

  • Include knockout/knockdown samples as negative controls where possible

  • Use pre-immune serum or isotype-matched control antibodies

  • Perform peptide competition assays to confirm specificity

What considerations should be made when selecting SULT1C2 antibodies for different applications?

When selecting SULT1C2 antibodies:

• Application-specific considerations:

  • Western blotting: Choose antibodies validated for denatured proteins

  • Immunohistochemistry: Select antibodies verified for specific fixation methods

  • Immunoprecipitation: Ensure antibodies can bind native protein conformations

  • ChIP: Select antibodies that function in crosslinked chromatin

• Epitope considerations:

  • For mitochondrial studies, ensure the epitope is accessible in this compartment

  • For detecting specific SULT1C2 variants, choose antibodies targeting unique regions

  • If studying protein-protein interactions, avoid antibodies that bind interaction interfaces

• Validation requirements:

  • Check if the antibody has been validated in your specific application

  • Review literature for successful use in similar experimental contexts

  • The SC-130274 antibody has been documented in SULT1C2 studies focusing on lung cancer

• Species reactivity:

  • For cross-species studies, verify reactivity with the target species

  • Consider sequence homology at the epitope region

  • Antibodies used in rat studies have successfully detected SULT1C2 in mitochondrial fractions

What are the emerging research directions for SULT1C2 antibodies?

Emerging areas for SULT1C2 antibody research:

• Therapeutic and diagnostic applications:

  • Development of SULT1C2 as a biomarker for cancer diagnosis and prognosis

  • SULT1C2 expression correlates with lung adenocarcinoma patient survival in smokers

  • SULT1C2 promotes growth and invasiveness in hepatocellular carcinoma

• Mitochondrial biology:

  • Further exploration of SULT1C2's role in mitochondrial membrane composition and function

  • Investigation of how cholesterol sulfate production affects mitochondrial physiology

  • Study of SULT1C2's protective effects against cellular stress conditions

• Personalized medicine approaches:

  • Development of tissue-based SULT1C2 assays for patient stratification

  • SULT1C2 promoter hypomethylation has been observed in Asian patients compared to Caucasians

  • Investigation of SULT1C2 as a potential therapeutic target

• Technological advances:

  • Development of more specific and sensitive antibodies for SULT1C2 detection

  • Creation of isoform-specific antibodies to distinguish between closely related SULT family members

  • Application of new imaging technologies to study SULT1C2 localization and dynamics

How can researchers best integrate SULT1C2 antibody data with other research methodologies?

For integrative research approaches:

• Multi-omics integration:

  • Combine antibody-based protein detection with transcriptomics and metabolomics

  • In HCC studies, SULT1C2 knockdown affected both gene expression and metabolite profiles

  • Correlate protein levels with enzymatic activity and functional outcomes

• Translational research strategies:

  • Connect basic research findings to clinical applications

  • Validate findings from cell culture models in patient samples

  • Develop standardized SULT1C2 detection methods for clinical laboratories

• Computational biology approaches:

  • Use structural biology to predict SULT1C2 interactions and functions

  • Develop models of how SULT1C2-mediated cholesterol sulfation affects mitochondrial membrane properties

  • Integrate protein expression data with patient outcomes for predictive modeling

• Collaborative research frameworks:

  • Establish standardized protocols for SULT1C2 detection across laboratories

  • Share validated antibody resources and controls

  • Develop common data repositories for SULT1C2 expression across tissues and disease states