SOAT2 Antibody

What is SOAT2 and what is its primary function in cellular metabolism?

SOAT2, also known as ACAT2 (Acyl-CoA:cholesterol acyltransferase-2), is an endoplasmic reticulum transmembrane enzyme belonging to the membrane-bound acyltransferase family and Sterol o-acyltransferase subfamily. Its primary function is to catalyze the formation of fatty acid-cholesterol esters, which are less soluble in membranes than free cholesterol . This esterification process plays a crucial role in protecting cells from the toxicity of excess free cholesterol.

SOAT2 is critical for lipoprotein assembly and dietary cholesterol absorption, showing particular substrate preference for oleoyl-CoA ((9Z)-octadecenoyl-CoA) and linolenoyl-CoA ((9Z,12Z,15Z)-octadecatrienoyl-CoA) . The enzyme facilitates the transport of cholesterol and triglycerides in the bloodstream by contributing to the formation of chylomicrons and very low-density lipoproteins (VLDL), thereby playing an essential role in maintaining cellular cholesterol homeostasis .

Where is SOAT2 predominantly expressed, and how does this correlate with its physiological functions?

SOAT2 demonstrates a tissue-specific expression pattern that directly correlates with its physiological functions:

• Predominantly expressed in enterocytes of the intestine and hepatocytes of the liver

• Localized in the endoplasmic reticulum of human intestinal cells

• Recently discovered to be overexpressed in regulatory T (Treg) cells from elderly patients with lung squamous cell carcinoma (LSCC)

This expression pattern aligns with SOAT2's roles in:

• Intestinal absorption and processing of dietary cholesterol

• Hepatic lipoprotein assembly and secretion

• Immune regulation, particularly in the context of aging and cancer

The differential expression in immune cells of elderly individuals suggests age-dependent regulation that may contribute to immune dysfunction and increased cancer susceptibility in the aging population .

What are the structural characteristics of SOAT2 and how do they relate to antibody binding sites?

SOAT2 is a 522 amino acid protein with a calculated molecular weight of approximately 60 kDa, although it is commonly observed at 46 kDa in experimental settings . This discrepancy between calculated and observed molecular weights should be considered when validating antibody specificity.

The protein contains multiple transmembrane domains as an ER-resident enzyme. When selecting antibodies, researchers should consider:

• Accessibility of epitopes - Some regions may be embedded in membranes or involved in protein-protein interactions

• Conservation across species - Important for cross-reactivity in comparative studies

• Functional domains - Antibodies targeting catalytic sites may have inhibitory effects

Available antibodies target different regions of the protein. For example, antibody ab230220 targets the N-terminal region (amino acids 1-150) , which may have different accessibility characteristics than antibodies targeting other regions.

What are the optimal conditions for detecting SOAT2 via Western blotting?

Based on validated protocols, the following conditions are recommended for optimal SOAT2 detection by Western blot:

It is essential to optimize these conditions for each specific experimental system. For instance, while the calculated molecular weight of SOAT2 is 60 kDa, it is typically observed at 46 kDa on Western blots . This discrepancy should be considered when interpreting results and may reflect post-translational processing or alternative splicing variants.

What are the key considerations for immunohistochemical detection of SOAT2?

For successful immunohistochemical detection of SOAT2, researchers should consider:

• Antigen Retrieval: Optimal results have been obtained using TE buffer pH 9.0, although citrate buffer pH 6.0 can serve as an alternative . This step is critical due to SOAT2's membrane localization.

• Antibody Dilution: A dilution range of 1:50-1:500 is recommended, though this should be optimized for each tissue type and fixation method .

• Validated Tissue Types: Human liver tissue and human small intestine tissue have been confirmed as positive controls . These tissues express high levels of SOAT2 naturally and serve as excellent reference samples.

• Fixation Effects: Overfixation can mask epitopes, particularly for membrane proteins like SOAT2. A fixation time of 24-48 hours in 10% neutral buffered formalin is generally appropriate.

• Detection System: Three-step detection systems may provide enhanced sensitivity compared to two-step methods, especially for low-abundance targets.

• Counterstaining: Light hematoxylin counterstaining allows for better visualization of tissue architecture without obscuring specific SOAT2 staining.

When interpreting IHC results, researchers should be aware of the characteristic cytoplasmic and perinuclear staining pattern typical of ER-localized proteins like SOAT2.

How can immunofluorescence be optimized for SOAT2 detection in cell culture systems?

For optimal immunofluorescence detection of SOAT2:

• Cell Fixation: 4% paraformaldehyde for 10-15 minutes at room temperature preserves cellular architecture while maintaining epitope accessibility.

• Permeabilization: 0.1-0.3% Triton X-100 for 5-10 minutes allows antibody access to intracellular antigens without excessive damage to membranes.

• Blocking: 5-10% normal serum (from the same species as secondary antibody) with 0.1% BSA reduces background staining.

• Antibody Dilution: A range of 1:50-1:500 is recommended , with HeLa cells serving as a positive control system .

• Co-staining Markers:

  • ER markers (e.g., calnexin, PDI) to confirm subcellular localization

  • Cell type-specific markers when working with heterogeneous populations

• Confocal Imaging: Z-stack imaging can help resolve the three-dimensional localization of SOAT2 within the ER network.

When imaging, researchers should look for the characteristic reticular pattern typical of ER proteins, with possible enrichment in specific ER domains involved in lipid metabolism.

How can SOAT2 antibodies be used to investigate age-related changes in regulatory T cells?

Recent research has identified SOAT2 as specifically overexpressed in regulatory T cells (Tregs) in elderly populations, with significant implications for immune aging and cancer progression . To investigate this phenomenon:

• Flow Cytometry Protocol:

  • Use multi-parameter panels including CD4, CD25, FOXP3 (for Treg identification), and SOAT2

  • Include age-matched controls when comparing young vs. elderly populations

  • Consider both frequency and mean fluorescence intensity (MFI) of SOAT2 expression

• Single-Cell Analysis Approach:

  • Combine SOAT2 antibody staining with single-cell RNA sequencing to correlate protein levels with transcriptional programs

  • Focus on cholesterol metabolism pathways and Treg functional markers

• Functional Correlation Studies:

  • Isolate SOAT2^high and SOAT2^low Treg populations using cell sorting

  • Assess suppressive capacity through conventional T cell proliferation assays

  • Evaluate expression of FOXP3 and other Treg functional markers

Research has demonstrated that SOAT2 overexpression in Treg cells promotes cholesterol metabolism by activating the SREBP2-HMGCR-GGPP pathway, enhancing Treg suppressor functions while reducing CD8+ T cell proliferation, migration, homeostasis and anti-tumor immunity . These findings highlight the potential of SOAT2 as a biomarker for immune senescence and a therapeutic target in age-related cancers.

What approaches can researchers use to study SOAT2's role in cancer progression and immune evasion?

To investigate SOAT2's role in cancer and immune evasion, researchers can implement these methodological approaches:

• Tumor Microenvironment Analysis:

  • Multiplex immunohistochemistry to simultaneously visualize SOAT2, FOXP3+ Tregs, CD8+ T cells, and tumor markers

  • Spatial transcriptomics to map SOAT2 expression patterns relative to immune infiltration zones

• In Vivo Models:

  • Compare tumor growth in young vs. aged mice with differential SOAT2 expression

  • Develop conditional knockout or overexpression models in Treg-specific manner

  • Assess immune infiltration through flow cytometry of tumor-infiltrating lymphocytes

• Clinical Correlation Studies:

  • Combine SOAT2 immunostaining with radiomics analysis in cancer patient cohorts

  • Assess correlation between SOAT2 expression, immune infiltration, and patient prognosis

Data from lung squamous cell carcinoma models indicates that high SOAT2 expression promotes tumor growth in aged mice . Analysis showed increased FOXP3-positive cells within the tumor microenvironment of SOAT2-high elder mice, but decreased FOXP3 expression in these cells, with SOAT2 expression inversely correlating with CD8+ T cell infiltration . These findings suggest complex mechanisms underlying SOAT2's role in tumor immune evasion.

How can researchers effectively evaluate SOAT2 inhibition as a potential therapeutic approach?

To evaluate SOAT2 inhibition as a therapeutic strategy, researchers should consider these methodological approaches:

• Target Validation Studies:

  • siRNA or CRISPR-based knockdown/knockout of SOAT2 in relevant cell types

  • Rescue experiments with exogenous SOAT2 expression to confirm specificity

  • Comparison of genetic vs. pharmacological inhibition approaches

• Pharmacological Inhibition Assessment:

  • Dose-response studies with SOAT2-specific inhibitors in cellular models

  • Evaluation of on-target effects through measurement of cholesterol ester formation

  • Assessment of off-target effects on related enzymes (e.g., SOAT1)

• Immune Function Evaluation:

  • Ex vivo treatment of Tregs with SOAT2 inhibitors followed by functional assays

  • In vivo administration of inhibitors with assessment of tumor-infiltrating lymphocyte profiles

  • Combination studies with established immunotherapies (e.g., checkpoint inhibitors)

The research indicating that SOAT2 overexpression activates the SREBP2-HMGCR-GGPP pathway, leading to enhanced Treg suppressor functions and reduced anti-tumor immunity , provides a mechanistic rationale for SOAT2 inhibition. This approach could potentially restore CD8+ T cell function and enhance anti-tumor responses, particularly in elderly cancer patients where SOAT2 overexpression has been documented.

How can researchers address discrepancies between expected and observed molecular weights for SOAT2?

The discrepancy between calculated (60 kDa) and observed (46 kDa) molecular weights of SOAT2 is a common challenge. Researchers can address this through:

• Verification Approaches:

  • Use multiple antibodies targeting different epitopes

  • Include positive control samples with known SOAT2 expression (HeLa cells, L02 cells)

  • Perform knockdown/knockout validation to confirm band specificity

• Technical Explanations to Consider:

  • Post-translational modifications affecting mobility

  • Proteolytic processing during sample preparation

  • Alternative splicing variants (SOAT2 has multiple isoforms with different enzymatic activities)

  • Protein conformation affecting SDS binding and gel migration

• Experimental Modifications:

  • Use gradient gels for better resolution

  • Modify sample preparation to minimize proteolysis

  • Include phosphatase or glycosidase treatments to assess post-translational modifications

When reporting results, researchers should explicitly mention both the expected and observed molecular weights, with appropriate citations to literature documenting similar observations.

What strategies can optimize detection sensitivity for low SOAT2 expression samples?

For samples with low SOAT2 expression, these optimization strategies can enhance detection sensitivity:

• Western Blot Enhancements:

  • Increase protein loading (up to 80-100 μg for tissue samples)

  • Use high-sensitivity chemiluminescent substrates

  • Employ signal enhancement systems (biotin-streptavidin amplification)

  • Extend primary antibody incubation to overnight at 4°C

  • Consider fluorescent secondary antibodies with infrared detection systems

• Immunohistochemistry/Immunofluorescence Improvements:

  • Implement tyramide signal amplification (TSA)

  • Use polymer-based detection systems rather than traditional ABC methods

  • Extend primary antibody incubation times (24-48 hours at 4°C)

  • Optimize antigen retrieval conditions (test multiple buffers and pH conditions)

• Pre-analytical Sample Enrichment:

  • Perform subcellular fractionation to enrich for ER membranes

  • Use immunoprecipitation to concentrate SOAT2 before detection

  • For cells, consider pre-treatment with cholesterol to potentially upregulate SOAT2

These approaches must be systematically optimized for each specific sample type, with appropriate positive and negative controls to confirm specificity.

How should researchers address cross-reactivity concerns when using SOAT2 antibodies?

To address potential cross-reactivity of SOAT2 antibodies:

• Validation Controls:

  • SOAT2 knockout/knockdown samples as negative controls

  • Peptide competition assays to confirm epitope specificity

  • Multiple antibodies targeting different epitopes for confirmation

  • Species-specific positive controls when working with non-human samples

• Technical Approaches:

  • Increase washing stringency (higher salt concentration, longer wash times)

  • Optimize blocking conditions (test different blocking agents: milk, BSA, serum)

  • Pre-adsorb antibodies with potential cross-reactive proteins/tissues

  • Use monoclonal antibodies for higher specificity in critical applications

• Analytical Considerations:

  • Sequence alignment analysis to identify potential cross-reactive proteins

  • Consider homology with SOAT1, which shares functional similarity

  • Be aware of tissue-specific expression patterns that may affect interpretation

Researchers should note that while polyclonal antibodies like 21852-1-AP show broader epitope recognition, potentially increasing sensitivity, monoclonal antibodies like sc-69837 may offer greater specificity for applications where cross-reactivity is a concern .

How can SOAT2 antibodies be utilized to investigate cholesterol metabolism in immune cell function?

Recent research reveals emerging roles for SOAT2 in immune cell function, particularly in the context of aging and cancer. To investigate these connections:

• Metabolic Profiling Approaches:

  • Combine SOAT2 immunophenotyping with lipidomics analysis

  • Correlate SOAT2 expression with cholesterol ester content in different immune cell subsets

  • Track metabolic pathway activation using phospho-specific antibodies for SREBP2 pathway components

• Functional Consequence Assessment:

  • Sort SOAT2^high vs. SOAT2^low immune populations for functional comparison

  • Combine with metabolic inhibitors to establish causality

  • Assess membrane fluidity and lipid raft composition in relation to SOAT2 activity

• Single-Cell Resolution Studies:

  • Implement imaging mass cytometry to correlate SOAT2 with multiple immune markers

  • Use proximity ligation assays to detect SOAT2 interactions with metabolic enzymes

  • Apply spatial transcriptomics to map SOAT2 activity zones in lymphoid tissues

Research has demonstrated that SOAT2 overexpression in Treg cells promotes cholesterol metabolism through the SREBP2-HMGCR-GGPP pathway, affecting both Treg suppressor functions and CD8+ T cell responses . This suggests a previously unrecognized link between cholesterol metabolism and immune regulation that may be particularly relevant in aging and cancer contexts.

What are the current limitations of SOAT2 antibodies for translational research, and how might they be addressed?

Current limitations of SOAT2 antibodies for translational research include:

• Specificity Challenges:

  • Limited validation in diverse tissue contexts

  • Potential cross-reactivity with related enzymes

  • Variability between antibody lots

  Addressing approach: Development of recombinant antibodies with defined epitope specificity and consistent production.

• Detection Sensitivity:

  • Suboptimal performance in formalin-fixed tissues

  • Limited sensitivity for detecting low expression levels

  • Variable performance across different applications

  Addressing approach: Implementation of signal amplification technologies and optimization of antigen retrieval methods specifically for SOAT2.

• Functional Correlation:

  • Most antibodies detect presence but not activity

  • Limited ability to distinguish between isoforms with different activities

  • Challenges linking expression to functional consequences

  Addressing approach: Development of conformation-specific or activity-state-specific antibodies that can distinguish active from inactive SOAT2.

• Cross-species Reactivity:

  • Variable performance across species complicates translational models

  • Limited validation in non-human primates and other model organisms

  Addressing approach: Systematic validation across species with conserved epitope targeting.

Ongoing development of more specific, sensitive, and functionally informative SOAT2 antibodies will be crucial for advancing translational research in this area.

What are the most promising future applications of SOAT2 antibodies in biomedical research?

Based on recent discoveries, several promising research directions for SOAT2 antibodies include:

• Biomarker Development:

  • Age-related immune dysfunction assessment

  • Stratification of cancer patients for immunotherapy responsiveness

  • Monitoring cholesterol metabolism dysregulation in metabolic diseases

• Therapeutic Target Validation:

  • Evaluation of SOAT2 inhibitors in cancer and metabolic disorders

  • Monitoring on-target engagement of therapeutic compounds

  • Assessment of combination approaches with existing therapies

• Fundamental Biology Exploration:

  • Investigation of SOAT2's role in immune cell senescence

  • Understanding tissue-specific regulation of cholesterol metabolism

  • Elucidating connections between lipid metabolism and immune function

The recent discovery of SOAT2's role in aged regulatory T cells and its impact on anti-tumor immunity opens new avenues for understanding how metabolic alterations affect immune function in aging and disease. SOAT2 antibodies will be essential tools for these investigations, enabling both basic research and translational applications aimed at exploiting this pathway for therapeutic benefit.

How might advances in antibody technology improve SOAT2 research in the coming years?

Emerging antibody technologies that may enhance SOAT2 research include:

• Advanced Recombinant Antibodies:

  • Site-specific conjugation for improved imaging and detection

  • Engineered fragments (Fabs, scFvs) for improved tissue penetration

  • Humanized antibodies for in vivo applications with reduced immunogenicity

• Functional Antibody Developments:

  • Activity-state specific antibodies that detect only active SOAT2

  • Conformation-selective antibodies that recognize specific protein states

  • Intrabodies capable of tracking SOAT2 in living cells

• Multiplexed Detection Systems:

  • Mass cytometry integration for simultaneous detection of SOAT2 with dozens of other markers

  • DNA-barcoded antibodies for ultra-high-parameter analysis

  • Spatial proteomics applications for analyzing SOAT2 in the tissue context

• Therapeutic Applications:

  • Antibody-drug conjugates targeting SOAT2 in pathological conditions

  • Bispecific antibodies linking SOAT2-expressing cells to immune effectors

  • Intracellular antibody delivery systems to modulate SOAT2 function