SETD6 Antibody, FITC conjugated

What is SETD6 and what are its primary biological functions?

SETD6 is a lysine methyltransferase that plays crucial roles in multiple cellular processes through methylation of various protein substrates. Its primary functions include regulation of NF-κB signaling pathways through monomethylation of RelA at lysine 310 (K310me1) , modulation of estrogen-responsive gene expression through association with estrogen receptor α (ERα) , and involvement in hippocampus-dependent learning and memory processes . SETD6 achieves these functions by forming protein complexes with transcriptional regulators such as HDAC1, MTA2, and TRRAP , thereby acting as either a transcriptional repressor or co-activator depending on the cellular context.

How does FITC conjugation affect SETD6 antibody applications?

FITC (Fluorescein isothiocyanate) conjugation of SETD6 antibodies provides direct fluorescent visualization capability without requiring secondary antibodies. This conjugation maintains antibody specificity while enabling several key applications:

• Direct immunofluorescence imaging of SETD6 localization

• Flow cytometry assessment of SETD6 expression levels

• Real-time visualization of SETD6 dynamics in live cell imaging

• Multiplex immunostaining when combined with other fluorophore-conjugated antibodies

The FITC fluorophore's excitation/emission profile (approximately 495nm/519nm) makes it compatible with standard FITC filter sets on most fluorescence microscopes, flow cytometers, and plate readers. For optimal results, samples should be protected from light exposure to prevent photobleaching of the FITC conjugate.

What experimental methods commonly employ SETD6 antibodies?

SETD6 antibodies are utilized in multiple experimental approaches:

• Immunoprecipitation to isolate SETD6 and associated protein complexes

• Western blotting to detect SETD6 protein levels and post-translational modifications

• Chromatin immunoprecipitation (ChIP) to identify SETD6 association with chromatin

• Immunofluorescence microscopy to visualize cellular localization

• Flow cytometry to quantify SETD6 expression levels

• Protein-protein interaction studies via co-immunoprecipitation and GST pulldown assays

For example, studies have used anti-SETD6 antibodies (Abbexa #005464) for protein co-immunoprecipitation to investigate SETD6 interactions with RelA (p65) and the subsequent effects on gene expression .

How can I validate the specificity of SETD6 antibody, FITC conjugated in my experimental system?

Validating SETD6 antibody specificity requires a multi-pronged approach:

• SETD6 knockdown/knockout controls: Compare FITC signal intensity between wild-type cells and those with SETD6 knockdown using siRNA or CRISPR/Cas9-mediated knockout . RNA-seq data from U251 control and SETD6 CRISPR knockout cells have shown significant differential expression of 2,104 genes, confirming successful SETD6 depletion .

• Peptide competition assay: Pre-incubate the FITC-conjugated SETD6 antibody with excess SETD6-specific peptide before application to samples. Specific binding should be blocked, resulting in signal reduction.

• Recombinant protein controls: Test antibody reactivity against purified recombinant SETD6 protein versus other SET domain-containing proteins.

• Cross-validation with multiple antibodies: Compare staining patterns using antibodies targeting different SETD6 epitopes.

• Western blot correlation: Confirm that immunofluorescence signal intensity correlates with protein levels detected by Western blot in the same samples.

For FITC-conjugated antibodies specifically, include an isotype control antibody (also FITC-conjugated) to establish background fluorescence levels.

What are the optimal fixation and permeabilization methods for SETD6 immunostaining with FITC-conjugated antibodies?

Optimal fixation and permeabilization methods depend on the cellular compartment where SETD6 is being studied:

For nuclear SETD6 detection:

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 for 10 minutes

• Block with 5% normal serum in PBS containing 0.1% Tween-20

For analysis of chromatin-bound SETD6:

• Pre-extract with 0.5% Triton X-100 in cytoskeleton buffer for 5 minutes on ice before fixation

• Fix with 4% paraformaldehyde

For cytoplasmic SETD6 detection:

• Use a milder fixation with 2% paraformaldehyde

• Permeabilize with 0.1% saponin instead of Triton X-100

Research has shown that SETD6 can localize to different cellular compartments, as demonstrated by cell fractionation studies separating cytoplasmic, nucleoplasmic, and chromatin-enriched fractions . Therefore, optimization of fixation methods is crucial for accurate detection of SETD6 in its various functional compartments.

How can I troubleshoot weak or non-specific signals when using FITC-conjugated SETD6 antibody?

When encountering weak or non-specific signals, consider these methodological adjustments:

For weak signals:

• Increase antibody concentration gradually (typically 1-5 μg/ml)

• Extend incubation time (overnight at 4°C)

• Optimize antigen retrieval methods if using fixed tissues

• Use signal amplification systems like tyramide signal amplification

• Ensure proper storage conditions to maintain FITC fluorescence (4°C, protected from light)

For non-specific signals:

• Increase blocking time and concentration (5-10% normal serum)

• Include 0.1-0.3% Triton X-100 in blocking and antibody dilution buffers

• Pre-absorb antibody with cell/tissue lysates from SETD6 knockout models

• Reduce antibody concentration

• Include additional washing steps with 0.1% Tween-20 in PBS

Studies have successfully used FITC-labeled peptides related to SETD6 substrates in cellular imaging by fixing cells and mounting with DAPI-containing medium , demonstrating the compatibility of FITC fluorescence with standard nuclear counterstaining protocols.

How can SETD6 antibodies be used to study the relationship between SETD6 and NF-κB signaling?

SETD6 antibodies are instrumental in investigating SETD6-mediated regulation of NF-κB signaling through several approaches:

• Detection of SETD6-mediated RelA methylation: Using specific antibodies against RelA K310me1 in conjunction with SETD6 antibodies to correlate SETD6 levels with RelA methylation status .

• ChIP-seq analysis: Using SETD6 antibodies to perform chromatin immunoprecipitation followed by sequencing to identify SETD6 occupancy at NF-κB target gene promoters .

• Co-immunoprecipitation studies: Employing SETD6 antibodies to pull down protein complexes and detect NF-κB subunits like RelA (p65) .

• SETD6 knockdown effects: Comparing NF-κB target gene expression in control versus SETD6-depleted cells using RT-qPCR and RNA-seq approaches .

Research has shown that SETD6 knockdown leads to increased expression of canonical NF-κB targets and cytokine production in various cell types including U2OS, THP-1, and primary mouse bone marrow-derived macrophages (mBMDM) cells. For instance, in response to TNF stimulation, SETD6-depleted cells showed increased production of secreted cytokines TNF and IL-6 compared to control siRNA-treated cells . This demonstrates the utility of SETD6 antibodies in elucidating signaling pathway alterations.

What is the significance of studying SETD6 localization in different cellular compartments?

Understanding SETD6 subcellular localization provides crucial insights into its diverse functions:

• Nuclear localization: Associated with its role in transcriptional regulation and chromatin modification. Nuclear SETD6 has been shown to interact with the estrogen receptor α and modulate estrogen-responsive gene expression .

• Chromatin association: SETD6 at chromatin can methylate histone H2AZ and RelA, leading to transcriptional repression through recruitment of GLP and subsequent H3K9 methylation .

• Cytoplasmic presence: May indicate roles in cytoplasmic signaling pathways and non-nuclear protein methylation.

Cell fractionation studies have demonstrated that SETD6 distributes between cytoplasmic, nucleoplasmic, and chromatin-enriched fractions . The proper detection of SETD6 in these compartments using FITC-conjugated antibodies requires appropriate cellular fractionation techniques followed by immunostaining or flow cytometry analysis.

How can SETD6 antibodies be used to investigate the role of SETD6 in learning and memory processes?

SETD6 antibodies have been employed to elucidate the role of SETD6 in hippocampus-dependent learning through several experimental approaches:

• Methylation state monitoring: Detecting changes in SETD6-mediated RELA-K310 methylation and H3K9me2 levels following contextual fear conditioning (CFC) .

• Protein complex identification: Using co-immunoprecipitation with SETD6 antibodies to identify learning-associated protein interactions in the hippocampus .

• Activity-dependent changes: Analyzing alterations in SETD6 levels and localization in response to learning activities.

• SETD6 knockdown effects: Examining consequences of SETD6 depletion on learning-associated transcriptional changes using RNA-seq .

Research has demonstrated that knockdown of SETD6 in the CA1 region of the hippocampus prevented learning-associated increases in RELA-K310me1 and H3K9me2 at 1 hour after contextual fear conditioning . RNA-seq analysis revealed robust differences in transcriptional profiles between control and SETD6-knockdown animals, with over 1,300 differentially expressed genes. Approximately 60% of all differentially expressed genes were overexpressed following SETD6 knockdown, while 72.9% of genes annotated for NF-κB regulation were upregulated . These findings highlight the essential role of SETD6 in orchestrating learning-induced transcriptional responses.

How can I design experiments to study SETD6 inhibition using FITC-conjugated tools?

Designing experiments to study SETD6 inhibition requires consideration of both detection methods and inhibition strategies:

• Peptide inhibitor approaches: Design FITC-labeled peptides based on known SETD6 substrates, such as RelA sequences containing K310 . These can serve dual purposes - as competitive inhibitors and as tools to visualize binding interactions.

• FRET-based enzyme assays: Develop assays using FITC-labeled substrate peptides and quencher-labeled product-specific antibodies to monitor SETD6 activity in real-time.

• Cellular uptake studies: Evaluate cellular penetration of potential SETD6 inhibitors using FITC-conjugated versions and monitoring by flow cytometry or microscopy .

• Displacement assays: Use FITC-labeled probes to screen for compounds that displace the fluorescent probe from the SETD6 binding pocket.

Studies have demonstrated the utility of FITC-labeled vp22-RelA peptides introduced into HEK293T and A549 cells transfected with mCherry-SETD6 for visualization of peptide-SETD6 interactions . A 15-amino acid synthetic peptide based on the RelA sequence containing the lysine residue methylated by SETD6 showed remarkable inhibitory effects on SETD6 methyltransferase activity .

What are the emerging roles of SETD6 in cancer biology and how can FITC-conjugated antibodies help investigate these functions?

SETD6 has emerging roles in cancer biology that can be investigated using FITC-conjugated antibodies:

• SETD6 in glioma progression: RNA-seq experiments with SETD6 CRISPR knockout U251 glioma cells revealed 2,104 differentially expressed genes, with significant enrichment of epithelial-mesenchymal transition (EMT) pathways among down-regulated genes . FITC-conjugated SETD6 antibodies can help visualize SETD6 expression patterns in different glioma grades and correlate with patient outcomes.

• SETD6 and TWIST1 interaction: SETD6 methylates TWIST1 on lysine-33 at chromatin, with expression levels correlating with poor survival in glioma . FITC-SETD6 antibodies can be used in co-localization studies with TWIST1 in tumor samples.

• SETD6 in estrogen-responsive cancers: SETD6 associates with estrogen receptor α (ERα) and functions as a co-activator of several estrogen-responsive genes . FITC-conjugated antibodies can track SETD6-ERα interactions in breast cancer models.

Gene correlation analysis of SETD6 mRNA levels and EMT-related transcription factors (SNAIL, SLUG, ZEB1, and TWIST1) in glioma patients revealed positive correlations with all these genes, suggesting that SETD6 might promote EMT in glioma . This provides rationale for using FITC-conjugated SETD6 antibodies to study EMT processes in tumor sections through co-localization with these transcription factors.

How can FITC-conjugated SETD6 antibodies be used in multiplexed imaging approaches?

FITC-conjugated SETD6 antibodies can be integrated into multiplexed imaging strategies through several approaches:

• Spectral multiplexing: Combine FITC-SETD6 antibodies with antibodies conjugated to spectrally distinct fluorophores (e.g., TRITC, Cy5, Alexa647) targeting other proteins of interest. This approach works well for co-localization studies of SETD6 with partners like HDAC1, MTA2, or RelA .

• Sequential multiplexing: Implement cyclic immunofluorescence methods where FITC-SETD6 antibody staining is performed, imaged, and then quenched/stripped before additional rounds of staining with other antibodies.

• Antibody-based signal amplification: Combine FITC-SETD6 antibodies with tyramide signal amplification systems for other targets to achieve higher multiplex capacity with minimal spectral overlap.

• Mass cytometry adaptation: While not utilizing the fluorescent properties of FITC, the same SETD6 antibody clones can be metal-tagged for mass cytometry (CyTOF) to achieve higher multiplexing capability.

Multiplexed approaches are particularly valuable for studying the various protein complexes that SETD6 forms. For instance, SETD6 has been shown to associate with HDAC1, MTA2, and TRRAP, but fails to associate with other NuRD complex subunits (CHD3 and RBAP46) or SIN3A complex components . Multiplexed imaging can help resolve these complex interaction patterns in different cellular contexts.

What controls should be included when using FITC-conjugated SETD6 antibodies in flow cytometry?

When employing FITC-conjugated SETD6 antibodies in flow cytometry, the following controls are essential:

• Unstained cells: To establish autofluorescence baseline

• Isotype control: FITC-conjugated antibody of the same isotype but irrelevant specificity

• SETD6 knockdown/knockout cells: Biological negative control

• Single-color controls: For compensation when performing multicolor flow cytometry

• Blocking peptide control: FITC-SETD6 antibody pre-incubated with excess SETD6 peptide

• Fixed versus unfixed comparison: To assess fixation effects on epitope accessibility

Additionally, when studying SETD6 in stimulation contexts like TNF or LPS treatment , include appropriate time-course controls to establish baseline SETD6 expression before stimulation.

How can I optimize SETD6 antibody performance in chromatin immunoprecipitation (ChIP) experiments?

Optimizing SETD6 antibody performance in ChIP experiments requires attention to several critical parameters:

• Crosslinking optimization: Test different formaldehyde concentrations (0.1-1%) and incubation times (5-15 minutes) to preserve SETD6-chromatin interactions while maintaining DNA accessibility.

• Sonication conditions: Optimize sonication to yield DNA fragments between 200-500bp while preserving SETD6 epitopes.

• Antibody amount: Titrate FITC-conjugated SETD6 antibody (typically 2-10 μg per ChIP reaction) and compare enrichment at known targets.

• Pre-clearing strategy: Include a pre-clearing step with protein A/G beads to reduce non-specific binding.

• Washing stringency: Test different salt concentrations in wash buffers to balance specificity and sensitivity.

Studies have successfully employed protein-protein ChIP approaches to detect SETD6 at chromatin, confirming its presence in chromatin-enriched fractions through biochemical separation techniques . These experiments revealed that SETD6 methylates RelA at chromatin, suggesting that optimization of ChIP conditions is crucial for detecting these chromatin-associated functions.

How should I interpret changes in SETD6 localization during cellular responses to stimuli?

Interpreting changes in SETD6 localization requires careful consideration of several factors:

• Baseline distribution: Establish the normal distribution of SETD6 between cytoplasmic, nucleoplasmic, and chromatin-enriched fractions in your cell type of interest .

• Stimulation-specific changes: Monitor redistribution following relevant stimuli like TNF treatment , estrogen exposure , or learning-related activities .

• Co-localization dynamics: Track changes in SETD6 association with known partners like RelA, HDAC1, or MTA2 .

• Functional correlation: Correlate localization changes with alterations in target gene expression, such as NF-κB-regulated genes .

Research has shown that SETD6 can repress NF-κB-driven inflammatory responses, with SETD6 knockdown increasing the production of secreted cytokines TNF and IL-6 in monocytic THP-1 cells exposed to TNF . The inverse correlation between SETD6 expression and NF-κB-linked inflammatory diseases suggests that monitoring SETD6 localization changes during inflammatory responses could provide insights into disease mechanisms.

How is SETD6 involved in the regulation of epithelial-mesenchymal transition in cancer?

SETD6 has emerged as a significant regulator of epithelial-mesenchymal transition (EMT) in cancer, particularly in glioma:

• Transcriptional profile impact: RNA-seq analysis of SETD6 CRISPR knockout cells identified 2,104 differentially expressed genes, with gene set enrichment analysis revealing significant enrichment of EMT pathways among down-regulated genes in SETD6-depleted cells .

• EMT transcription factor correlation: Gene correlation analysis demonstrated positive correlations between SETD6 mRNA levels and key EMT-related transcription factors (SNAIL, SLUG, ZEB1, and TWIST1) in glioma patients .

• TWIST1 methylation: SETD6 methylates TWIST1 on lysine-33 at chromatin, potentially regulating its activity in promoting EMT and cancer progression .

• Survival correlation: SETD6 expression levels correlate with poor survival in glioma patients, suggesting its role as a potential prognostic marker .

These findings suggest that FITC-conjugated SETD6 antibodies could be valuable tools for investigating the spatial and temporal dynamics of SETD6 involvement in EMT processes, potentially identifying new therapeutic targets for aggressive cancers.

What recent advances have been made in understanding SETD6's role in learning and memory formation?

Recent research has revealed critical roles for SETD6 in learning and memory processes:

• Learning-induced methylation: SETD6 mediates learning-associated increases in RELA-K310me1 and H3K9me2 in the hippocampus following contextual fear conditioning .

• Transcriptional regulation: Knockdown of SETD6 results in widespread transcriptional changes in the hippocampus, with over 1,300 differentially expressed genes .

• NF-κB pathway modulation: Approximately 73% of genes annotated for NF-κB regulation were upregulated following SETD6 knockdown, suggesting SETD6 normally represses NF-κB-dependent transcription during memory consolidation .

• Ontology enrichment: Reactome pathway analysis of SETD6-regulated genes identified significant enrichment of ontologies relevant to learning and memory, including "Transmission across Chemical Synapses" and "Neurotransmitter Receptors and Postsynaptic Signal Transmission" .

These findings establish SETD6 as a critical player in orchestrating gene expression programs during memory consolidation, potentially opening new avenues for addressing cognitive disorders.

What are the emerging therapeutic applications targeting SETD6 methyltransferase activity?

Emerging therapeutic approaches targeting SETD6 include:

• Peptide inhibitors: A 15-amino acid synthetic peptide based on the RelA sequence containing the lysine residue methylated by SETD6 has demonstrated remarkable inhibitory effects on SETD6 methyltransferase activity . This represents a promising approach for developing targeted SETD6 inhibitors.

• Anti-inflammatory applications: Given SETD6's role in repressing NF-κB-driven inflammatory responses , modulation of SETD6 activity could have therapeutic potential for inflammatory conditions.

• Cancer therapy approaches: The correlation between SETD6 expression and poor survival in glioma patients , along with its role in promoting EMT, suggests SETD6 inhibitors could have applications in cancer treatment.

• Cognitive enhancement strategies: Understanding SETD6's role in learning and memory may lead to approaches for enhancing cognitive function or addressing memory disorders.

Peptide-based strategies offer several advantages for targeting SETD6, including high target specificity and selectivity with relatively low cytotoxicity . FITC-conjugated peptides further enable visualization of cellular uptake and distribution, facilitating the development and optimization of therapeutic approaches.

What are the most promising future applications of FITC-conjugated SETD6 antibodies in epigenetic research?

FITC-conjugated SETD6 antibodies hold significant promise for advancing several areas of epigenetic research:

• Live-cell imaging: Developing cell-permeable FITC-conjugated SETD6 antibody fragments or nanobodies could enable real-time tracking of SETD6 dynamics during cellular responses to stimuli.

• Super-resolution microscopy: Applying techniques like STORM or PALM with FITC-SETD6 antibodies to resolve nanoscale associations with chromatin structures and transcriptional machinery.

• Single-cell analysis: Combining FITC-SETD6 antibodies with single-cell technologies to map heterogeneity in SETD6 expression and localization across diverse cell populations.

• Proximity labeling approaches: Integrating FITC-SETD6 antibodies with proximity labeling methods (BioID, APEX) to comprehensively map the SETD6 interaction network in different cellular contexts.

The consistent finding that SETD6 functions at the intersection of multiple critical cellular pathways—from NF-κB signaling to estrogen receptor function to EMT regulation —underscores the value of developing increasingly sophisticated tools to study this multifunctional methyltransferase.

How might improved SETD6 detection methodologies enhance our understanding of epigenetic regulation?

Advanced SETD6 detection methodologies could transform our understanding of epigenetic regulation in several ways:

• Temporal resolution: Development of rapid, sensitive SETD6 detection methods would enable tracking of dynamic changes in SETD6 activity during fast cellular responses, such as the kinetics of inflammatory signaling .

• Multi-omics integration: Combining FITC-SETD6 antibody-based approaches with genomic, transcriptomic, and proteomic methods could provide integrated views of SETD6's role in coordinating multiple layers of regulation.

• Substrate-specific detection: Engineering antibodies that specifically recognize SETD6-methylated substrates (beyond RelA K310me1) would expand our understanding of SETD6's regulatory scope.

• In vivo imaging: Adapting FITC-SETD6 antibodies for in vivo imaging could bridge cellular findings to organismal physiology and disease models.

The discovery that SETD6 controls more than 1,300 genes upon knockdown and significantly affects EMT pathways in cancer suggests that improved detection methodologies will likely uncover additional regulatory networks under SETD6 control.