SAT1 Antibody, HRP conjugated

What is SAT1 and why is it significant in research?

SAT1 (Spermidine/spermine N1-acetyltransferase 1) is the key regulatory enzyme in polyamine catabolism, catalyzing acetylation of spermidine or spermine to generate N1-acetyl spermidine or N1-acetyl spermine, and N1, N12-diacetylspermine. This enzyme is particularly significant in research because its cellular level is normally extremely low but can be rapidly induced by various stimuli, including polyamines, polyamine analogs, toxic chemicals, certain drugs, and growth factors. Recent research has revealed its involvement in cancer mechanisms, particularly in enabling anchorage independence and peritoneal metastasis in ovarian cancer .

What is the molecular weight range for SAT1 in experimental detection?

While the calculated molecular weight of SAT1 is approximately 20 kDa, the observed molecular weight typically ranges between 15-25 kDa in Western blot analyses. This variation may be attributable to post-translational modifications or different isoforms. Cell Signaling Technology's SAT1 antibody specifically detects a band at 18 kDa .

What are the advantages of using HRP-conjugated antibodies for SAT1 detection?

HRP (Horseradish Peroxidase) conjugation provides several methodological advantages for SAT1 detection:

• Eliminates the need for secondary antibody incubation, reducing experimental time and potential cross-reactivity

• Enables direct visualization in Western blotting and immunohistochemistry through enzymatic conversion of substrates

• Provides enhanced sensitivity in enzyme-linked immunosorbent assays (ELISA)

• Reduces background signal by eliminating potential non-specific binding from secondary antibodies

• Allows for more precise quantification in colorimetric and chemiluminescent detection systems

What are the optimal dilution ranges for SAT1 antibody across different applications?

Optimal dilution ranges vary by application and specific antibody format:

Western Blotting (WB):

• Unconjugated SAT1 antibodies: 1:500-1:3000 (Proteintech) or 1:1000 (Cell Signaling)

• HRP-conjugated SAT1 antibodies: 1:1000-1:6000

Immunohistochemistry (IHC):

• 1:50-1:500 for paraffin-embedded sections

Immunofluorescence (IF)/Immunocytochemistry (ICC):

• 1:50-1:500

Flow Cytometry (Intracellular):

• 0.25 μg per 10^6 cells in a 100 μl suspension

These ranges should be optimized for specific sample types and experimental conditions .

What buffer conditions are optimal for SAT1 antibody stability and performance?

For optimal SAT1 antibody stability and performance:

Storage Buffer: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 is recommended for long-term storage.

Working Buffers for Applications:

• Western Blotting: TBS-T (Tris-buffered saline with 0.1% Tween-20) with 5% non-fat dry milk or BSA

• IHC: PBS or TBS with optimal antigen retrieval using TE buffer (pH 9.0) or citrate buffer (pH 6.0)

• Immunofluorescence: PBS with 1% BSA and 0.3% Triton X-100

For HRP Conjugation:

• 10-50mM amine-free buffer (e.g., HEPES, MES, MOPS, phosphate) pH 6.5-8.5

• Avoid buffers containing nucleophilic components like primary amines and thiols

• Avoid sodium azide as it inhibits HRP activity

How should antigen retrieval be performed for SAT1 detection in tissue sections?

For optimal antigen retrieval in SAT1 immunohistochemistry:

Primary Recommendation: TE buffer at pH 9.0

• Deparaffinize and rehydrate tissue sections

• Place slides in TE buffer (10mM Tris, 1mM EDTA, pH 9.0)

• Heat using a pressure cooker or microwave to 95-100°C for 15-20 minutes

• Allow to cool slowly to room temperature (approximately 20 minutes)

• Wash thoroughly in PBS before proceeding with blocking and antibody incubation

Alternative Method: Citrate buffer at pH 6.0

• Heat slides in 10mM citrate buffer (pH 6.0) for 15-20 minutes

• Follow cooling and washing steps as above

These retrieval methods have been validated with mouse brain tissue but should be optimized for specific tissue types .

How can SAT1 antibodies be effectively used to study its noncanonical role in histone modification?

Recent research has uncovered SAT1's noncanonical role in histone acetylation, particularly H3K27ac. To study this function:

• Chromatin Immunoprecipitation (ChIP) Approach:

  • Perform dual ChIP with anti-SAT1 and anti-H3K27ac antibodies

  • Use ChIP-seq to identify genomic regions where SAT1 and H3K27ac co-localize

  • Analyze enrichment at mitosis-regulating genes and chromosome organization pathways

• Co-Immunoprecipitation Protocol:

  • Cross-link proteins using formaldehyde (1%, 10 minutes)

  • Lyse nuclei and sonicate chromatin

  • Immunoprecipitate with SAT1 antibody

  • Probe for H3 interaction by Western blot

  • Use HRP-conjugated SAT1 antibodies for direct detection

• In Vitro Acetylation Assay:

  • Incubate recombinant His-H3 protein with flag-SAT1

  • Add acetyl-CoA (labeled if quantification needed)

  • Detect acetylation using anti-H3K27ac antibody

  • Validate specificity by including SAT1 enzyme inhibitor (like ginkgolide B)

This methodological approach will help elucidate SAT1's role in regulating H3K27ac marks within genes required for mitosis regulation and chromosome segregation .

What experimental design is recommended to study SAT1 induction under hypoxic conditions?

To investigate SAT1 induction under hypoxic conditions:

• Cell Culture Setup:

  • Compare attached versus detached cell cultures (e.g., using ultra-low attachment plates)

  • Include normoxic and hypoxic conditions (1-2% O₂) in a controlled chamber

  • Use hypoxia probe (e.g., pimonidazole) to confirm hypoxic conditions

• Molecular Analysis Workflow:

  • Extract RNA for RT-qPCR and protein for Western blot at multiple time points (0, 6, 12, 24 hours)

  • Perform ChIP assay using anti-HIF-1α antibody to detect binding at the SAT1 promoter

  • Include HIF-1α knockdown controls to confirm the regulatory mechanism

  • Use luciferase reporter assay with wild-type and mutated HRE sequence

• Detection Methods:

  • Western blot with HRP-conjugated SAT1 antibody for direct visualization

  • Immunofluorescence to detect cellular localization shifts during hypoxia

  • Flow cytometry for quantitative analysis of SAT1 expression at the single-cell level

This comprehensive approach allows investigation of the temporal dynamics of SAT1 induction under hypoxic conditions, particularly in the context of detached cells as seen in peritoneal metastasis models .

How can researchers distinguish between SAT1's canonical polyamine metabolism function and its noncanonical histone modification role?

To differentiate between these distinct SAT1 functions:

• Site-Directed Mutagenesis Approach:

  • Generate SAT1 mutants (e.g., K166A mutant) that maintain one function while disrupting the other

  • Validate mutant function using in vitro enzyme assays for both:

    • Polyamine acetylation activity (using spermine substrate)

    • Histone acetylation activity (using H3 substrate)

• Functional Separation Protocol:

  • Measure cellular polyamine levels using HPLC or LC-MS/MS

  • Assess H3K27ac levels by Western blot and ChIP-seq

  • Compare effects of wild-type SAT1 versus mutants on both readouts

  • Test recovery of function with specific mutants in SAT1-depleted cells

• Inhibition Studies Design:

  • Use ginkgolide B (uncompetitive inhibitor, Ki ≈ 24.18 μM)

  • Monitor differential effects on polyamine metabolism versus histone acetylation

  • Combine with tryptophan fluorescence binding assay to confirm binding mechanisms

  • Test SAT1 Y163F mutant to validate inhibitor binding site

This methodological approach allows researchers to parse the distinct biochemical functions of SAT1 and their respective contributions to cellular phenotypes .

What are the potential causes and solutions for weak or no signal when using HRP-conjugated SAT1 antibodies?

When encountering detection issues with HRP-conjugated SAT1 antibodies:

Potential Causes and Solutions:

Remember that SAT1 levels are typically very low in normal conditions but are induced by specific stimuli, which may necessitate experimental induction before detection .

How can researchers validate the specificity of their SAT1 antibody results?

To ensure specificity and validity of SAT1 antibody results:

• Positive and Negative Controls:

  • Positive: Use HEK-293, K-562, or HeLa cells (known to express SAT1)

  • Negative: Include SAT1 knockdown/knockout samples (siRNA, shRNA, or CRISPR)

  • Recombinant protein: Include purified SAT1 protein as standard

• Cross-Validation Protocol:

  • Compare results from multiple antibody clones/sources

  • Verify using different detection methods (WB, IHC, IF)

  • Confirm with non-antibody techniques (RT-qPCR, mass spectrometry)

• Technical Validation Methods:

  • Peptide competition assay: Pre-incubate antibody with blocking peptide

  • Molecular weight verification: Confirm 15-25 kDa band (specifically ~18-20 kDa)

  • Expression pattern consistency: Compare with literature reports

• Advanced Validation for HRP-Conjugated Antibodies:

  • Compare direct HRP-conjugated antibody with unconjugated primary + HRP-secondary approach

  • Perform enzyme activity control to verify HRP functionality

  • Include isotype control antibody with HRP conjugation

These comprehensive validation steps ensure experimental results accurately reflect SAT1 biology rather than artifacts .

What precautions should be taken when using SAT1 antibodies in multiplex assays?

For successful multiplex assays incorporating SAT1 antibodies:

• Cross-Reactivity Prevention:

  • Select antibodies raised in different host species

  • Verify no cross-reactivity between secondary detection systems

  • Use highly cross-adsorbed secondary antibodies

• Signal Separation Strategies:

  • For fluorescent multiplexing: Choose fluorophores with minimal spectral overlap

  • For chromogenic IHC: Use sequential rather than simultaneous detection

  • For chemiluminescent WB: Strip and reprobe or use spectrally distinct substrates

• HRP-Specific Considerations:

  • When using multiple HRP-conjugated antibodies, sequential detection is required

  • Complete HRP inactivation between rounds (3% H₂O₂, 10 minutes)

  • Consider tyramide signal amplification (TSA) for enhanced sensitivity and distinct signals

• Technical Optimization:

  • Titrate antibody concentrations individually before multiplexing

  • Optimize fixation conditions compatible with all target epitopes

  • Include single-stain controls to verify signal specificity

These methodological approaches will enable reliable multiplexed detection of SAT1 alongside other targets of interest .

How does SAT1 expression correlate with cancer progression and metastasis?

Recent research has revealed important correlations between SAT1 and cancer progression:

• Expression Analysis Findings:

  • SAT1 expression is significantly associated with metastasis in serous ovarian cancer

  • SAT1 is strongly induced in detached cancer cells compared to attached monolayer cells

  • Hypoxia inducible factor-1α (HIF-1α) directly regulates SAT1 expression via hypoxia response element (HRE) in its promoter

• Functional Significance:

  • SAT1 enables anchorage-independent survival of cancer cells

  • SAT1 depletion increases DNA damage (γH2AX) and apoptosis in detached cells

  • SAT1's noncanonical function in histone acetylation (particularly H3K27ac) regulates genes involved in mitosis and chromosome segregation

• Clinical Correlations:

  • Higher SAT1 expression correlates with poorer patient outcomes

  • Cancer cells from ascites (detached) show higher SAT1 dependence than primary tumors

  • SAT1 inhibition (e.g., with ginkgolide B) selectively affects viability of cancer cells from ascites

These findings suggest SAT1 as a potential biomarker and therapeutic target in metastatic disease, particularly in peritoneal metastasis of ovarian cancer .

What are the most effective protocols for conjugating HRP to SAT1 antibodies while maintaining specificity and sensitivity?

For optimal HRP conjugation to SAT1 antibodies:

• Pre-Conjugation Preparation:

  • Start with high-purity antibody (>95% by SDS-PAGE)

  • Buffer exchange into amine-free buffer (10-50mM HEPES, MES, MOPS, or phosphate, pH 6.5-8.5)

  • Antibody concentration should be 0.5-5.0 mg/ml for optimal conjugation

• Conjugation Protocol Using LYNX Rapid Kit:

  • Add 1μl modifier reagent per 10μl antibody and mix

  • Add mixture to lyophilized HRP and gently resuspend

  • Incubate at room temperature (20-25°C) for 3 hours or overnight

  • Add 1μl quencher reagent per 10μl original antibody

  • Incubate 30 minutes before use

• Molar Ratio Optimization:

  • Optimal antibody:HRP ratio ranges from 1:1 to 1:4

  • For 100μg HRP, use 100-400μg SAT1 antibody

  • For 1mg HRP, use 1-4mg SAT1 antibody

• Post-Conjugation Validation:

  • Compare activity to unconjugated antibody using known positive samples

  • Verify maintained specificity using knockdown controls

  • Test storage stability at various timepoints

This methodology ensures high conjugation efficiency with 100% antibody recovery while maintaining specificity and sensitivity .

How can researchers differentiate between the various molecular forms and post-translational modifications of SAT1 protein?

To distinguish between SAT1 variants and modifications:

• Gel Electrophoresis Approach:

  • Use high-resolution SDS-PAGE (12-15% gels) to separate closely spaced bands

  • Consider Phos-tag™ gels to detect phosphorylated forms

  • Perform 2D gel electrophoresis to separate based on both pI and molecular weight

• Western Blot Protocol for Modification Detection:

  • Probe with modification-specific antibodies after SAT1 immunoprecipitation

  • Use specific inhibitors of post-translational modifications to characterize bands

  • Compare patterns in different cellular contexts (attached vs. detached, normoxic vs. hypoxic)

• Mass Spectrometry Analysis:

  • Perform immunoprecipitation with SAT1 antibody

  • Analyze by LC-MS/MS for comprehensive identification of modifications

  • Use targeted MS approaches for specific modification sites

• Functional Characterization:

  • Compare enzymatic activity of different SAT1 forms

  • Assess cellular localization by fractionation and immunofluorescence

  • Evaluate interaction partners by co-immunoprecipitation

These methodological approaches provide a comprehensive analysis of SAT1 variants and modifications, which can have distinct regulatory and functional implications .

How can SAT1 antibodies be effectively employed in chromatin immunoprecipitation studies to investigate its role in histone modification?

For effective ChIP studies of SAT1's role in histone modification:

• Dual ChIP-seq Experimental Design:

  • Perform ChIP with both anti-SAT1 and anti-H3K27ac antibodies

  • Analyze co-localization patterns genome-wide

  • Focus analysis on promoters of mitosis-regulating genes

• Sequential ChIP (Re-ChIP) Protocol:

  • First immunoprecipitate with anti-SAT1 antibody

  • Elute complexes under non-denaturing conditions

  • Perform second immunoprecipitation with anti-H3K27ac

  • This identifies genomic regions bound by both proteins simultaneously

• ChIP-qPCR Validation:

  • Design primers for key target genes (e.g., genes involved in mitotic cell cycle, chromosome organization)

  • Compare enrichment patterns between control and SAT1-depleted cells

  • Include appropriate positive controls (known H3K27ac-enriched regions) and negative controls

• Integrated Analysis Approach:

  • Correlate ChIP-seq data with RNA-seq from the same experimental conditions

  • Perform Gene Ontology and pathway enrichment analysis

  • Validate findings with ginkgolide B treatment to inhibit SAT1 activity

This comprehensive approach enables detailed characterization of SAT1's chromatin interactions and their functional consequences .

What strategies can resolve inconsistencies between antibody-based detection of SAT1 and functional enzymatic assays?

To address discrepancies between antibody detection and enzyme activity:

• Combined Detection Strategy:

  • Perform parallel analysis of SAT1 protein levels (by Western blot with HRP-conjugated antibody) and enzyme activity (using labeled substrates)

  • Include recombinant SAT1 standards for calibration

  • Compare results across multiple experimental conditions

• Epitope-Function Relationship Analysis:

  • Map antibody epitopes relative to functional domains

  • Test if antibody binding affects enzyme activity

  • Use multiple antibodies targeting different regions of SAT1

• Post-Translational Modification Assessment:

  • Evaluate if modifications affect antibody recognition versus enzyme activity

  • Use phosphatase or deacetylase treatment prior to antibody detection

  • Compare native versus denatured protein detection

• Methodological Reconciliation Protocol:

  • Normalize enzyme activity to absolute protein quantity determined by mass spectrometry

  • Develop a correction factor for specific experimental conditions

  • Consider the effect of protein complexes on epitope accessibility versus enzyme activity

This systematic approach helps reconcile potentially discordant results between antibody-based detection and functional assays, providing a more complete understanding of SAT1 biology .

How can advanced imaging techniques be optimized to study SAT1 subcellular localization during cellular stress responses?

For advanced imaging of SAT1 localization during stress:

• Super-Resolution Microscopy Protocol:

  • Use STORM or PALM techniques for nanoscale resolution

  • Label SAT1 with photoactivatable or photoswitchable fluorophores

  • Co-label with organelle markers to precisely define localization

• Live-Cell Imaging Strategy:

  • Generate SAT1-GFP/RFP fusion proteins with careful validation of functionality

  • Design microfluidic systems for real-time stress application during imaging

  • Include hypoxia chamber setup for oxygen-dependent regulation studies

• Multi-Channel 3D Confocal Approach:

  • Perform z-stack imaging for complete cellular volume

  • Co-stain with markers for:

    • Nuclear compartments (e.g., chromatin, nucleoli)

    • Cytoplasmic organelles (mitochondria, ER, Golgi)

    • Stress-induced structures (stress granules, P-bodies)

• Quantitative Image Analysis Pipeline:

  • Develop automated segmentation for subcellular compartments

  • Measure co-localization coefficients (Pearson's, Mander's)

  • Track dynamic changes in localization over time after stress induction

  • Correlate localization changes with functional readouts

This comprehensive imaging approach reveals the dynamic subcellular redistribution of SAT1 during stress responses, providing insights into its context-dependent functions .

What statistical approaches are most appropriate for analyzing variability in SAT1 detection across different tissue samples?

For robust statistical analysis of SAT1 detection variability:

• Data Normalization Methods:

  • Use housekeeping proteins as internal controls (β-actin, GAPDH, tubulin)

  • Consider tissue-specific reference genes for more accurate normalization

  • Apply global normalization methods for large-scale analyses

• Appropriate Statistical Tests:

  • For normally distributed data: ANOVA with post-hoc tests (Tukey, Bonferroni)

  • For non-parametric data: Kruskal-Wallis with Dunn's multiple comparison

  • For paired samples: Paired t-test or Wilcoxon signed-rank test

  • For correlations: Pearson's or Spearman's correlation coefficients

• Variance Component Analysis:

  • Distinguish biological from technical variability

  • Implement mixed-effects models to account for nested experimental designs

  • Use coefficient of variation (CV) to quantify relative variability

• Advanced Analytical Approaches:

  • Linear regression models to identify factors influencing SAT1 detection

  • Principal component analysis to identify patterns in multivariate data

  • Hierarchical clustering to identify sample subgroups with similar SAT1 patterns

These statistical approaches enable robust interpretation of SAT1 detection data, accounting for various sources of variability .

How should researchers interpret discrepancies in SAT1 molecular weight across different experimental platforms?

To properly interpret SAT1 molecular weight variations:

• Technical Factor Analysis:

  • Gel percentage effects: Higher percentage gels (12-15%) provide better resolution in 15-25 kDa range

  • Running buffer composition: Tris-glycine versus Tris-tricine systems affect migration patterns

  • Sample preparation: Denaturation conditions can affect apparent molecular weight

  • Molecular weight standards: Different standards can yield different size estimates

• Biological Explanation Framework:

  • Post-translational modifications: Phosphorylation, acetylation, or ubiquitination

  • Alternative splicing: Different SAT1 isoforms

  • Proteolytic processing: N or C-terminal cleavage during sample preparation

  • Species differences: Comparing human (calculated 20 kDa) versus mouse or rat SAT1

• Verification Protocol:

  • Mass spectrometry analysis for definitive molecular weight determination

  • Use of multiple antibodies targeting different epitopes

  • Treatment with phosphatases or deglycosylation enzymes

  • Comparison with recombinant protein standards

• Reporting Standards:

  • Document precise experimental conditions

  • Report observed molecular weight as a range (e.g., 15-25 kDa)

  • Include positive control samples with known molecular weight

This systematic approach helps researchers interpret and reconcile molecular weight variations in SAT1 detection across different experimental systems .

What emerging technologies might enhance the sensitivity and specificity of SAT1 detection beyond current antibody-based methods?

Several emerging technologies show promise for improved SAT1 detection:

• Proximity Ligation Assay (PLA) Applications:

  • Allows detection of protein-protein interactions in situ

  • Can detect SAT1 interactions with H3 or other partners with single-molecule sensitivity

  • Protocol: Use two primary antibodies (anti-SAT1 and anti-H3), followed by species-specific secondary antibodies with attached oligonucleotides that form a circle when in close proximity, enabling rolling circle amplification and fluorescent detection

• CRISPR-Based Tagging Systems:

  • CRISPR-mediated knockin of small epitope tags or fluorescent proteins

  • Enables detection of endogenous SAT1 without antibody variability issues

  • Application: Generate cell lines with tagged endogenous SAT1 for consistent detection

• Aptamer-Based Detection:

  • Develop RNA or DNA aptamers with high specificity for SAT1

  • Advantages include chemical stability, reproducibility, and no batch variation

  • Methodology: Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to identify SAT1-specific aptamers

• Mass Cytometry (CyTOF):

  • Uses metal-tagged antibodies and time-of-flight mass spectrometry

  • Enables highly multiplexed single-cell analysis without spectral overlap issues

  • Implementation: Develop metal-conjugated SAT1 antibodies for integration into CyTOF panels

These technologies offer significant advantages for sensitivity, specificity, and multiplexing capabilities in SAT1 detection for future research applications .

How might advances in structural biology inform the development of more specific SAT1 antibodies and inhibitors?

Recent structural insights can guide development of improved SAT1 research tools:

• Structure-Guided Antibody Development:

  • Target highly specific epitopes based on structural analysis

  • Design antibodies that distinguish between SAT1's canonical and noncanonical functions

  • Methodology: Utilize information about SAT1's interaction with H3 (particularly residues around Y140, R142, and K166) to develop antibodies that selectively recognize specific functional states

• Rational Inhibitor Design:

  • Improve upon ginkgolide B (Ki ≈ 24.18 μM) using structure-activity relationships

  • Target the uncompetitive inhibition mechanism through the SAT1:H3 complex

  • Focus on interactions with SAT1 Y163 and H3 G35 sites

  • Application: Develop inhibitors with improved potency, selectivity, and pharmacological properties

• Conformation-Specific Detection Tools:

  • Develop antibodies that recognize specific SAT1 conformational states

  • Distinguish between polyamine-bound, histone-bound, and free states

  • Technical approach: Use of structural information to design antibodies against conformation-specific epitopes

• Multimodal Structural Analysis:

  • Combine X-ray crystallography, cryo-EM, and molecular dynamics simulations

  • Generate comprehensive structural models of SAT1 in different functional contexts

  • Application: Use integrated structural information to develop more precise research tools

These structure-guided approaches will enable development of next-generation research tools with enhanced specificity for different aspects of SAT1 biology .

What methodological adaptations would be required to study SAT1's role in human clinical samples versus established cell lines?

Transitioning from cell lines to clinical samples requires several methodological adaptations:

• Sample Preparation Protocol Modifications:

  • Tissue fixation optimization: Compare FFPE versus frozen tissue preservation

  • Antigen retrieval: Test multiple conditions (TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Protein extraction: Adapt protocols for limited sample quantities

  • Cell type heterogeneity: Include microdissection or single-cell approaches

• Detection Sensitivity Enhancement:

  • Signal amplification: Implement tyramide signal amplification for IHC

  • Multiplex staining: Combine SAT1 with cell type markers for contextual information

  • Digital pathology: Use computational image analysis for quantitative assessment

  • Ultrasensitive WB: Apply capillary-based automated Western systems

• Clinical Context Integration:

  • Design paired analysis of primary tumors and metastases

  • Include adjacent normal tissue controls

  • Correlate with patient metadata (treatment history, outcome measures)

  • Develop tissue microarrays for high-throughput analysis

• Validation Requirements:

  • Antibody validation in human tissues (positive/negative controls)

  • Cross-platform verification (IHC, WB, IF)

  • Correlation with orthogonal measures (mRNA levels, enzyme activity)

  • Implementation of standard operating procedures for reproducibility