retsat Antibody

What is RETSAT and what cellular functions does it serve?

RETSAT (Retinol Saturase) is an oxidoreductase enzyme that catalyzes the saturation of all-trans-retinol to all-trans-13,14-dihydroretinol. In humans, the canonical protein consists of 610 amino acid residues with a molecular mass of approximately 66.8-69 kDa . RETSAT is primarily localized in the endoplasmic reticulum (ER) but also exhibits nuclear localization .

The protein serves multiple biological functions including:

• Regulation of retinol metabolism as a member of the Carotenoid/retinoid oxidoreductase protein family

• Potential tumor suppression, with lower expression correlating with worse clinical outcomes in multiple cancer types

• Modulation of immune infiltration in various cancer types

• Stabilization of mitotic chromosome segregation in pluripotent stem cells

• Protection against oxidative stress in certain cellular contexts

RETSAT expression is regulated by various transcription factors including PPARα in liver, PPARγ in adipose tissue, and FOXO1 in primary hepatocytes . Its expression positively correlates with obesity and liver steatosis .

What are the key considerations for selecting a RETSAT antibody for immunodetection?

When selecting a RETSAT antibody for immunodetection, researchers should consider several critical factors to ensure experimental success:

Reactivity: Verify that the antibody recognizes RETSAT from your species of interest. Available antibodies have confirmed reactivity with human, mouse, and rat RETSAT . Cross-reactivity information should be carefully reviewed, especially when working with non-human models.

Applications: Different antibodies are validated for specific applications. Current RETSAT antibodies are validated for Western blot (WB), ELISA, Flow Cytometry (FACS), and immunofluorescence (IF) . For example, Proteintech's antibody (16895-1-AP) is validated for ELISA , while NSJ Bio's antibody (RQ7777) is validated for WB (0.5-1 μg/ml), FACS (1-3 μg/million cells), and direct ELISA (0.1-0.5 μg/ml) .

Antibody format: Consider whether you need a monoclonal or polyclonal antibody. Polyclonal antibodies may provide higher sensitivity but potentially lower specificity compared to monoclonals. Several suppliers offer polyclonal antibodies raised in rabbits , while some laboratories have developed monoclonal antibodies .

Immunogen information: Review the specific region of RETSAT used as the immunogen. For instance, NSJ Bio's antibody was raised against E. coli-derived recombinant human RETSAT (amino acids E35-K597) , which may impact epitope recognition and functional blocking potential.

Validation data: Examine the validation data provided by manufacturers, including western blot images showing the expected molecular weight band (approximately 67 kDa) .

How should I optimize RETSAT antibody dilution for Western blot applications?

Optimizing RETSAT antibody dilution for Western blot requires systematic testing to balance signal strength with background reduction:

Initial dilution range: Begin with the manufacturer's recommended dilution. For example, NSJ Bio recommends 0.5-1 μg/ml for their RETSAT antibody . If no specific recommendation exists, start with standard dilutions (1:500 to 1:2000).

Titration approach: Perform a titration experiment using a series of antibody dilutions (e.g., 1:500, 1:1000, 1:2000, 1:5000) against a positive control sample with known RETSAT expression. Human liver samples or certain cell lines with confirmed RETSAT expression serve as appropriate positive controls .

Blocking optimization: Test different blocking agents (5% non-fat milk, 5% BSA) to determine which provides optimal signal-to-noise ratio. Phosphate-buffered blocking solutions with trehalose (as used in some commercial antibody formulations) may help preserve antibody activity .

Exposure time adjustment: When using chemiluminescent detection, capture multiple exposure times to identify the optimal signal without saturation. RETSAT typically appears as a band at approximately 67 kDa .

Background reduction: If high background persists, try increased washing steps, higher dilutions of secondary antibody, or addition of 0.1% Tween-20 to wash buffers.

Document all optimization steps in your laboratory notebook for reproducibility of results and protocol refinement.

How can I effectively use RETSAT antibodies to study its role in cancer progression?

Investigating RETSAT's role in cancer progression requires a multifaceted approach combining antibody-based techniques with molecular and cellular analyses:

Tissue microarray analysis: Utilize RETSAT antibodies for immunohistochemistry on cancer tissue microarrays to evaluate expression patterns across tumor types and stages. Research indicates RETSAT is downregulated in multiple cancer types, with lower expression correlating with worse clinical outcomes .

Correlation with immune infiltration: Combine RETSAT immunostaining with immune cell markers to analyze relationships between RETSAT expression and tumor immune microenvironment. Studies have shown positive correlations between RETSAT expression and infiltration levels of B cells (r = 0.597), CD8+ T cells (r = 0.422), CD4+ T cells (r = 0.671), macrophages (r = 0.478), neutrophils (r = 0.744), and dendritic cells (r = 0.837) in skin cutaneous melanoma (SKCM) .

Methylation analysis: Complement protein detection with promoter methylation studies, as RETSAT promoter hypermethylation contributes to its decreased expression in tumor tissues . Use bisulfite sequencing or methylation-specific PCR alongside antibody detection to correlate methylation status with protein levels.

Functional studies: Employ RETSAT antibodies in combination with RETSAT knockdown or overexpression models to monitor changes in cell proliferation. Research shows that RETSAT knockdown promotes, while overexpression inhibits, cell proliferation in mouse embryonic fibroblasts (MEFs) and B16 cells in vitro .

Co-immunoprecipitation: Use RETSAT antibodies for co-IP experiments to identify interaction partners involved in cancer-related pathways. Some RETSAT antibodies have been validated for immunoprecipitation applications, requiring approximately 3 μg per experiment .

What are the best practices for troubleshooting non-specific binding when using RETSAT antibodies?

Non-specific binding is a common challenge when working with antibodies. For RETSAT antibodies, implement these troubleshooting strategies:

Validation with knockout controls: Whenever possible, include RETSAT knockout or knockdown samples as negative controls. Research groups have developed RETSAT knockout mouse embryonic stem cells that serve as excellent specificity controls .

Blocking optimization: Test different blocking agents beyond the standard 5% milk or BSA. For RETSAT detection, adding 1-2% normal serum from the species of your secondary antibody can reduce non-specific binding.

Antibody titration refinement: Non-specific binding often results from excessive antibody concentration. Create a more detailed titration series around your preliminary optimal dilution (e.g., if 1:1000 seemed optimal, test 1:800, 1:1000, 1:1200).

Pre-absorption controls: Pre-incubate your RETSAT antibody with recombinant RETSAT protein (when available) to confirm specificity. The signal should be significantly reduced or eliminated in pre-absorbed samples compared to non-absorbed controls.

Cross-reactivity assessment: If your experiment involves multiple proteins, particularly other retinoid pathway enzymes, verify whether your RETSAT antibody cross-reacts with structurally similar proteins. Most commercial RETSAT antibodies state "no cross-reactivity with other proteins" in their documentation .

Alternative antibody evaluation: Compare results using antibodies from different vendors or those targeting different epitopes of RETSAT. For example, some antibodies target the E35-K597 region , while others may target different domains.

How can I effectively use RETSAT antibodies to investigate its role in oxidative stress responses?

To study RETSAT's involvement in oxidative stress responses, implement these methodological approaches:

Cell viability assays with RETSAT modulation: Use RETSAT antibodies to confirm knockdown or overexpression efficiency before subjecting cells to oxidative stress inducers like tert-butyl hydroperoxide (tert-BHP). Studies show RETSAT expression positively correlates with cellular sensitivity to oxidative stress inducers .

ROS detection alongside RETSAT immunostaining: Combine RETSAT immunofluorescence with fluorescent ROS indicators like DCFDA to correlate RETSAT expression levels with ROS generation at the single-cell level.

Lipid peroxidation measurement: Implement thiobarbituric acid-reactive substances (TBARS) assays to quantify lipid peroxidation in cells with different RETSAT expression levels following oxidative stress. Research has employed this approach using RETSAT antibodies to confirm expression levels in experimental and control groups .

Sub-cellular localization during stress: Utilize immunofluorescence with RETSAT antibodies to track potential changes in protein localization during oxidative stress. Given RETSAT's dual localization in the ER and nucleus, this could reveal stress-induced translocation.

Rescue experiments: In RETSAT-depleted cells, perform add-back experiments with all-trans-13,14-dihydroretinol (the product of RETSAT enzymatic activity) to determine if the metabolite can rescue oxidative stress phenotypes. Use RETSAT antibodies to confirm the absence of protein in these experimental systems .

Co-immunoprecipitation under stress conditions: Apply RETSAT antibodies in co-IP experiments before and after oxidative stress induction to identify stress-specific interaction partners that may explain RETSAT's role in stress responses.

How can I integrate RETSAT antibody data with genomic and transcriptomic analyses to understand its role in tumor immunity?

Integrating RETSAT protein expression data with genomic and transcriptomic analyses requires sophisticated multi-omics approaches:

Copy number variation correlation: Analyze the relationship between RETSAT copy number variation (CNV) and protein expression using RETSAT antibodies. Research shows RETSAT CNV significantly correlates with infiltrating levels of B cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells .

Multi-parameter flow cytometry: Develop flow cytometry panels incorporating RETSAT antibodies alongside markers for immune cell subpopulations and activation states. This approach can reveal correlations between RETSAT expression and specific immune cell phenotypes at the single-cell level.

Spatial transcriptomics integration: Combine RETSAT immunohistochemistry with spatial transcriptomics to correlate protein expression with transcriptional programs in the tumor microenvironment. This can identify spatial relationships between RETSAT-expressing cells and infiltrating immune cells.

Correlation with immunomodulatory molecules: Analyze relationships between RETSAT expression and immunostimulators/immunoinhibitors. Research has shown that RETSAT expression positively correlates with immunostimulators and negatively correlates with immunoinhibitors in skin cutaneous melanoma .

RNA-seq validation: Use RETSAT antibodies to stratify samples for RNA-seq analysis, allowing correlation between protein levels and transcriptional profiles. This approach can identify pathways co-regulated with RETSAT that may explain its immunomodulatory effects.

Methylation pattern correlation: Integrate RETSAT protein quantification with promoter methylation analysis to understand epigenetic regulation of RETSAT in immune contexts. Research indicates promoter hypermethylation contributes to decreased RETSAT expression in tumors .

What methodological approaches can resolve contradictory data between RETSAT's reported tumor suppressor and oncogenic roles?

Resolving contradictory findings regarding RETSAT's role in cancer requires sophisticated experimental designs:

Context-specific expression analysis: Use RETSAT antibodies to profile expression across diverse tissue and tumor types under standardized conditions. RETSAT may have context-dependent functions, acting as a tumor suppressor in some cancers while promoting survival in others .

Isoform-specific detection: Develop experimental protocols using antibodies targeting specific RETSAT isoforms. With up to two different isoforms reported , differential functions may explain contradictory findings.

Subcellular fractionation studies: Implement rigorous fractionation followed by western blotting with RETSAT antibodies to determine localization patterns in different cell types. RETSAT's function may differ depending on its cytoplasmic versus nuclear localization .

Cell-cycle dependency analysis: Combine RETSAT immunostaining with cell cycle markers to determine if its function varies throughout the cell cycle. Research shows specific roles in mitotic chromosome loading , which may be distinct from its functions in non-dividing cells.

Post-translational modification mapping: Use phospho-specific antibodies or mass spectrometry following RETSAT immunoprecipitation to identify post-translational modifications that might switch its function between tumor-promoting and tumor-suppressing roles.

Conditional knockout models: Generate tissue-specific or inducible RETSAT knockout systems to examine temporal and spatial requirements. Use RETSAT antibodies to confirm knockdown efficiency and correlate with phenotypic outcomes in different contexts.

Interactome analysis in different contexts: Perform co-immunoprecipitation with RETSAT antibodies across different cell types, followed by mass spectrometry to identify context-specific interaction partners that might explain divergent functions.

How can RETSAT antibodies be applied to investigate its chromosome loading function in pluripotent stem cells?

Investigating RETSAT's chromosome loading function in pluripotent stem cells requires specialized immunofluorescence and biochemical approaches:

Immunofluorescence during mitosis: Optimize RETSAT antibody protocols for co-staining with chromosome markers at various mitotic stages. Research demonstrates RETSAT localizes to mitotic chromosomes specifically in stemness-positive cells like ESCs and iPSCs .

Chromatin immunoprecipitation (ChIP): Adapt RETSAT antibodies for ChIP applications to identify specific chromosomal regions bound by RETSAT. This requires careful optimization of crosslinking conditions and sonication parameters for mitotic chromosomes.

Co-localization with cohesin/condensin: Perform multi-color immunofluorescence with RETSAT antibodies alongside cohesin/condensin components like Smc1a, Smc3, and Nudcd2. Research confirms that RETSAT interacts with these components and affects their loading onto chromosomes .

Proximity ligation assays (PLA): Implement PLA using RETSAT antibodies paired with antibodies against chromosome stability factors to visualize and quantify protein-protein interactions in situ with nanometer resolution.

Live cell imaging: Combine RETSAT immunofluorescence with live cell imaging techniques to track dynamic chromosome loading during differentiation. RETSAT localizes to chromosomes specifically in stemness-positive cells and this pattern changes during differentiation .

Correlation with pluripotency factors: Perform co-immunostaining for RETSAT and pluripotency factors like Nanog to establish relationships between stemness and RETSAT's chromosome loading function. Research suggests RETSAT displays active viewpoints similar to Nanog in embryonic stem cells .

Sequential ChIP (Re-ChIP): Develop protocols using RETSAT antibodies in conjunction with antibodies against chromosome structural proteins to identify genomic regions where these factors co-occupy.

What technical considerations are important when using RETSAT antibodies for detecting its involvement in adaptation to hypoxic conditions?

Studying RETSAT's role in hypoxia adaptation requires specialized technical approaches:

Hypoxia chamber optimization: When staining cells grown under hypoxic conditions, optimize fixation protocols to prevent reoxygenation artifacts. Perform fixation directly in the hypoxia chamber before exposing cells to atmospheric oxygen.

Mutation-specific antibody application: Consider developing or obtaining antibodies that specifically recognize the Q247R RETSAT variant identified in hypoxia adaptation . This mutation represents a glutamine to arginine switch that enhances heart function under hypoxic conditions.

Spatial co-expression analysis: Implement multi-color immunofluorescence with RETSAT antibodies alongside hypoxia markers like HIF-1α to establish spatial relationships in tissue sections from hypoxia models.

Correlation with oxidative stress markers: Combine RETSAT immunostaining with detection of oxidative stress markers, as research indicates RETSAT modulates responses to oxidative stress , which is closely linked to hypoxia adaptation.

Time-course experiments: Design experiments tracking RETSAT expression and localization at multiple timepoints during hypoxic exposure. This can reveal temporal dynamics of RETSAT's involvement in hypoxia adaptation.

Interaction with RNA helicases: Optimize co-immunoprecipitation protocols using RETSAT antibodies to detect interactions with RNA helicases like DDX39B, which has been implicated in RETSAT's role in promoting stalled fork restarting under replicative stress .

Co-staining with DNA damage markers: Implement protocols combining RETSAT antibodies with markers of DNA damage response, as RETSAT has been implicated in UV-induced DNA damage response and genomic stability .

How should researchers evaluate and validate commercial RETSAT antibodies for experimental reproducibility?

Ensuring reproducibility with RETSAT antibodies requires systematic validation steps:

Positive control selection: Use tissues with known high RETSAT expression, such as liver samples, as positive controls . Cell lines with confirmed RETSAT expression can also serve as controls.

Multiple antibody comparison: Test antibodies from different vendors targeting distinct epitopes. Current commercial options include antibodies targeting E35-K597 and others with different epitope targets.

Knockout/knockdown validation: Generate RETSAT knockdown samples using validated siRNAs (e.g., those targeting mouse RETSAT exon 2 or exon 9) to confirm antibody specificity. RETSAT knockout mouse embryonic stem cells also serve as excellent negative controls .

Western blot molecular weight verification: Confirm that detected bands appear at the expected molecular weight of approximately 67 kDa . Be aware that post-translational modifications or different isoforms may result in slight variations.

Cross-reactivity testing: If working across species, validate the antibody in each species of interest. While many antibodies claim reactivity with human, mouse, and rat RETSAT , actual performance may vary.

Lot-to-lot consistency assessment: When receiving new antibody lots, perform side-by-side comparisons with previous lots to ensure consistent performance. Document lot numbers and performance characteristics.

Alternative detection methods: Confirm RETSAT expression using orthogonal techniques such as RT-qPCR alongside antibody-based detection to ensure consistency between protein and mRNA levels.

What are the most effective fixation and antigen retrieval methods for RETSAT immunostaining in different tissue types?

Optimizing fixation and antigen retrieval for RETSAT immunostaining varies by tissue type:

Liver tissue processing: Since RETSAT is highly expressed in liver , this tissue requires careful optimization. For paraffin sections, test both 10% neutral buffered formalin and Bouin's fixative, with the former typically providing better antigen preservation for RETSAT.

Heat-induced versus enzymatic retrieval: Compare heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) versus Tris-EDTA (pH 9.0) for RETSAT antibodies. For many nuclear proteins like RETSAT, which has nuclear localization in some contexts , alkaline pH buffers often provide superior results.

Pluripotent stem cell fixation: For ESCs and iPSCs, where RETSAT shows specific chromosome loading , test 4% paraformaldehyde fixation with varying durations (10-20 minutes) to preserve chromosome structure while enabling antibody access.

Permeabilization optimization: Compare different permeabilization agents (0.1-0.5% Triton X-100, 0.1-0.5% Saponin, or 100% methanol) to maximize nuclear antigen accessibility while preserving membrane-associated RETSAT pools.

Fresh-frozen versus FFPE comparison: If working with archival tissues, compare RETSAT detection in matched fresh-frozen and FFPE samples to establish optimal protocols for retrospective studies.

Sequential immunostaining: When combining RETSAT detection with other markers, determine whether sequential or simultaneous antibody incubation provides better results. For co-localization with chromosome markers, sequential staining often reduces background.

Blocking serum selection: Test normal sera from different species (goat, donkey, horse) to identify optimal blocking conditions that minimize non-specific binding while preserving specific RETSAT signal.

How can researchers integrate RETSAT antibody-based studies with metabolomic analyses of retinoid pathways?

Integrating RETSAT antibody detection with retinoid pathway metabolomics requires careful experimental design:

Correlation between protein levels and metabolite quantification: Use RETSAT antibodies to quantify protein expression in parallel with LC-MS/MS detection of retinoid metabolites, particularly all-trans-retinol and all-trans-13,14-dihydroretinol (RETSAT's substrate and product) .

Substrate availability experiments: Design experiments varying retinol concentration while monitoring RETSAT protein levels using antibody detection. This can reveal feedback mechanisms between substrate availability and enzyme expression.

Enzymatic activity correlation: Develop assays measuring RETSAT enzymatic activity and correlate with protein levels detected by antibodies. This can identify post-translational regulatory mechanisms where protein abundance doesn't directly correlate with activity.

Subcellular fractionation with metabolite tracking: Use RETSAT antibodies to confirm successful fractionation of ER membranes, where RETSAT primarily localizes , then analyze retinoid metabolites in these fractions to establish compartment-specific metabolism.

13,14-dihydroretinol rescue experiments: In cells with RETSAT knockdown confirmed by antibody detection, supplement with synthetic all-trans-13,14-dihydroretinol to determine which phenotypes can be rescued by the metabolite alone versus those requiring the protein itself .

Retinoid synthesis inhibition studies: Combine pharmacological inhibition of upstream retinoid synthesis with RETSAT protein detection to establish how pathway perturbation affects RETSAT expression and localization.

Mass spectrometry imaging integration: In tissue sections, correlate RETSAT immunohistochemistry with mass spectrometry imaging of retinoid metabolites to establish spatial relationships between enzyme expression and metabolite distribution.

How might RETSAT antibodies be applied in studying its potential as a biomarker for pluripotent stem cell quality?

Exploring RETSAT as a PSC quality biomarker requires specialized antibody applications:

High-throughput screening protocols: Develop automated immunofluorescence protocols using RETSAT antibodies to rapidly assess chromosome loading in PSC cultures. Research suggests RetSat could serve as an effective biomarker for assessing pluripotent stem cell quality .

Correlation with genomic stability markers: Implement co-staining protocols with RETSAT antibodies and markers of genomic instability (micronuclei, chromosome bridges) to establish RETSAT's predictive value for PSC genomic integrity .

Flow cytometry sorting: Optimize intracellular staining protocols for RETSAT to enable flow cytometry-based selection of high-quality PSCs with proper chromosome loading.

Single-cell analysis: Develop protocols integrating RETSAT immunofluorescence with single-cell transcriptomics to correlate protein expression heterogeneity with transcriptional signatures of pluripotency and differentiation potential.

Live cell imaging: Adapt antibody fragments or nanobodies against RETSAT for live-cell imaging applications to monitor chromosome loading dynamics during PSC culture without fixation.

Differentiation tracking: Implement time-course studies using RETSAT antibodies to track changes in chromosome loading during directed differentiation. This could identify critical windows where RETSAT expression predicts differentiation outcomes.

Comparative analysis across reprogramming methods: Use RETSAT antibodies to compare chromosome loading efficiency in iPSCs generated through different reprogramming methods, potentially identifying optimal protocols for generating genomically stable iPSCs.

What methodological advances could improve the detection of RETSAT's interaction with cohesin/condensin components?

Advanced methodologies for studying RETSAT-cohesin/condensin interactions include:

Proximity-dependent biotin labeling: Adapt BioID or TurboID approaches using RETSAT as the bait protein to identify proximity interactors on chromosomes, validating findings with antibodies against Smc1a, Smc3, and Nudcd2 .

Super-resolution microscopy: Implement STORM or PALM imaging using fluorophore-conjugated RETSAT antibodies to visualize nanoscale chromosome loading patterns relative to cohesin/condensin components with resolution below the diffraction limit.

In situ proximity ligation assays: Optimize PLA protocols using RETSAT antibodies paired with antibodies against cohesin/condensin components to visualize and quantify direct interactions on chromosomes in fixed cells.

FRET-based interaction analysis: Develop fluorescence resonance energy transfer (FRET) approaches using fluorophore-conjugated antibodies against RETSAT and cohesin/condensin components to detect direct interactions in fixed cells.

ChIP-sequencing integration: Combine ChIP-seq using RETSAT antibodies with existing cohesin/condensin ChIP-seq datasets to identify genomic regions where these factors co-localize, potentially revealing functional interaction domains.

Quantitative chromosome proteomics: Implement Chromatin Mass Spectrometry (ChroP-MS) using RETSAT antibodies for immunoprecipitation of chromosome-associated complexes, followed by mass spectrometry to identify and quantify associated proteins including cohesin/condensin components.

Split-pool barcoding: Develop combinatorial indexing immunofluorescence approaches using RETSAT antibodies alongside cohesin/condensin markers to achieve high-throughput single-cell profiling of chromosome loading patterns.

What are the most promising future research directions for RETSAT antibody applications?

The future of RETSAT antibody applications spans several promising directions:

Therapeutic target validation: As RETSAT emerges as a potential tumor suppressor , antibodies will be crucial for validating its expression in patient samples and correlating with treatment responses in preclinical models.

Biomarker development: The potential of RETSAT as a quality control marker for pluripotent stem cells represents an exciting avenue for clinical translation of regenerative medicine .

Understanding retinoid metabolism in disease: RETSAT antibodies will help elucidate the role of altered retinoid metabolism in conditions ranging from metabolic disorders to cancer, potentially identifying new therapeutic opportunities.

Chromosome stability mechanisms: Further investigation into RETSAT's role in chromosome stability using advanced antibody-based imaging techniques may reveal fundamental insights into genome maintenance mechanisms .

Epigenetic regulation: The observed promoter methylation of RETSAT in cancer suggests opportunities to explore epigenetic targeting strategies, with antibodies serving as essential tools for validating target engagement .

Immune modulation: The strong correlation between RETSAT expression and immune cell infiltration in tumors opens avenues for exploring its potential role in immunotherapy response prediction .

Hypoxia adaptation mechanisms: RETSAT's implicated role in hypoxia adaptation presents opportunities for understanding fundamental mechanisms of cellular adaptation to environmental stress, with antibodies enabling spatial and temporal tracking of RETSAT dynamics .