SETD6 Antibody, HRP conjugated

What is SETD6 and what cellular functions does it regulate?

SETD6 is a 473 amino acid protein that belongs to the SETD6 family and contains one SET domain. It functions as a protein lysine N-methyltransferase that specifically monomethylates "Lys-310" of the NFκB p65 subunit of the NFκB complex. This methylation effectively down-regulates NFκB transcription factor activity by rendering the NFκB p65 subunit inert . Through this mechanism, SETD6 attenuates NFκB p65-driven transcriptional programs, including inflammatory responses in primary immune cells. The SETD6-initiated lysine-methylation signaling cascade restrains activation of NFκB-mediated inflammatory responses across diverse cell types .

Recent research has also implicated SETD6 in controlling cell adhesion, migration, and cancer cell survival, making it a protein of interest in cancer research, particularly in B-cell Acute Lymphoblastic Leukemia (B-ALL) . SETD6 interacts with numerous proteins involved in metabolic processes, muscle contraction, and protein folding as revealed by proteomic studies .

What applications are HRP-conjugated SETD6 antibodies suitable for?

HRP-conjugated SETD6 antibodies are specifically designed for applications requiring direct enzymatic detection without secondary antibody steps. Based on available data, these antibodies are suitable for:

• Western Blotting (WB): Provides direct detection of SETD6 protein without requiring secondary antibody incubation .

• ELISA: Particularly valuable for quantitative measurement of SETD6 protein levels .

• Immunohistochemistry (IHC): Used for detection of SETD6 in tissue samples .

• Immunocytochemistry (ICC): For cellular localization studies of SETD6 .

The HRP conjugation provides enhanced sensitivity and streamlines experimental workflows by eliminating secondary antibody incubation steps, which is particularly advantageous in time-sensitive protocols .

What are the key specifications researchers should consider when selecting SETD6 antibodies?

When selecting SETD6 antibodies for experimental use, researchers should evaluate:

• Antibody Specificity: Target region specificity (e.g., AA 1-74 vs. AA 310-338) .

• Host Species: Typically rabbit for many available SETD6 antibodies .

• Clonality: Polyclonal antibodies offer broader epitope recognition .

• Species Reactivity: Most commonly Human, though some antibodies react with Mouse and Rat SETD6 .

• Conjugation: HRP-conjugated for direct detection, or unconjugated for use with secondary antibodies .

• Purification Method: Protein G purification yields >95% purity in many commercial antibodies .

• Storage Requirements: Typically stable for 1 year at 2-8°C from date of receipt .

How can researchers validate the specificity of SETD6 antibodies?

Validating SETD6 antibody specificity is essential for reliable experimental results. Researchers should implement the following methodological approaches:

• Positive and Negative Control Samples:

  • Positive controls: Cell lines known to express SETD6 (e.g., K562 cells) .

  • Negative controls: SETD6 knockout cell lines or SETD6-silenced cells through siRNA/shRNA.

• Western Blot Band Confirmation:

  • Verify the molecular weight of detected bands (SETD6 is 473 amino acids).

  • Compare with recombinant SETD6 protein to confirm band position.

• Antibody Pre-absorption Test:

  • Pre-incubate antibody with recombinant SETD6 protein before application.

  • Signal should be reduced or eliminated if antibody is specific.

• Cross-Validation with Multiple Antibodies:

  • Compare results from antibodies targeting different SETD6 epitopes (e.g., N-terminal vs. C-terminal) .

  • Consistent results across different antibodies suggest higher specificity.

• Immunoprecipitation Combined with Mass Spectrometry:

  • Perform IP using the SETD6 antibody followed by MS analysis.

  • Specific antibodies should predominantly pull down SETD6 and known interacting proteins .

What are optimal protocols for using HRP-conjugated SETD6 antibodies in Western blotting?

For optimal Western blot results with HRP-conjugated SETD6 antibodies, researchers should follow this methodological workflow:

• Sample Preparation:

  • Lyse cells in RIPA buffer (50 mM Tris–HCl, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mM DTT and protease inhibitor cocktail) .

  • Include phosphatase inhibitors if studying SETD6 phosphorylation status.

• Gel Electrophoresis and Transfer:

  • Separate 20-50 μg of protein lysate on 10-12% SDS-PAGE.

  • Transfer to PVDF membrane (preferred over nitrocellulose for better protein retention).

• Blocking:

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

  • For phospho-specific detection, use 5% BSA exclusively.

• Primary Antibody Incubation:

  • Dilute HRP-conjugated SETD6 antibody (optimal working dilution should be determined by the investigator, but typically 1:1000-1:5000) .

  • Incubate overnight at 4°C with gentle rocking.

• Washing and Detection:

  • Wash membrane 3-5 times with TBST (5 minutes each).

  • Proceed directly to detection using ECL substrate (no secondary antibody needed).

  • For low abundance targets, consider using enhanced chemiluminescence substrates.

• Stripping and Reprobing (if needed):

  • Use mild stripping buffer (200 mM glycine, 0.1% SDS, 1% Tween 20, pH 2.2).

  • Always validate complete stripping before reprobing.

How should researchers troubleshoot weak or non-specific signals with SETD6 antibodies?

When encountering weak or non-specific signals with SETD6 antibodies, implement the following systematic troubleshooting strategies:

• For Weak Signals:

  • Increase antibody concentration incrementally (e.g., from 1:2000 to 1:1000).

  • Extend primary antibody incubation (overnight at 4°C instead of 1-2 hours).

  • Use enhanced detection substrates for HRP-conjugated antibodies.

  • Increase protein loading amount (50-100 μg).

  • Check sample degradation by staining for housekeeping proteins.

  • Ensure SETD6 is expressed in your experimental system (verify with RT-PCR).

• For Non-specific Signals:

  • Increase blocking stringency (5-10% blocking agent).

  • Add 0.1-0.5% Tween-20 to antibody dilution buffer.

  • Optimize antibody concentration (excessive antibody causes non-specific binding).

  • Use freshly prepared buffers and reagents.

  • Perform additional washing steps with increased TBST volume.

  • Try alternative HRP-conjugated SETD6 antibodies targeting different epitopes .

• For Background Issues:

  • Use fresh membranes and blocking agents.

  • Ensure complete blocking (minimum 1 hour at room temperature).

  • Perform extensive washing between steps.

  • Filter all buffers before use.

  • Check for contamination in the ECL substrate.

How can SETD6 antibodies be utilized to study SETD6's role in NFκB signaling pathways?

SETD6 antibodies provide valuable tools for investigating SETD6's regulatory role in NFκB signaling through the following methodological approaches:

• Co-Immunoprecipitation Studies:

  • Use SETD6 antibodies to pull down SETD6 protein complexes from cell lysates.

  • Western blot analysis of the immunoprecipitated material for NFκB p65 or other NFκB complex components to confirm interaction .

  • Verify through reciprocal IP with NFκB p65 antibodies followed by SETD6 detection.

• Chromatin Immunoprecipitation (ChIP) Assays:

  • Use SETD6 antibodies to identify genomic regions where SETD6 is recruited.

  • Combine with ChIP for NFκB p65 to determine co-occupancy at specific promoters.

  • Sequential ChIP (re-ChIP) can definitively demonstrate co-occupancy of SETD6 and NFκB.

• Immunofluorescence Co-localization:

  • Use fluorescently labeled SETD6 antibodies alongside NFκB component antibodies.

  • Track localization changes following inflammatory stimuli (e.g., TNFα treatment).

  • Quantify nuclear translocation of NFκB in the presence/absence of SETD6.

• Methylation-Specific Detection:

  • Combine SETD6 antibodies with antibodies specific for methylated Lys-310 of NFκB p65.

  • Monitor changes in methylation status following SETD6 overexpression or knockdown.

  • Correlate methylation status with NFκB transcriptional activity through reporter assays.

• Proximity Ligation Assay (PLA):

  • Visualize and quantify direct interactions between SETD6 and NFκB components in situ.

  • Assess how these interactions change under different cellular conditions or stimuli.

These methodologies allow researchers to establish the mechanistic details of how SETD6-mediated methylation regulates NFκB activity, revealing its role in attenuating NFκB-driven transcriptional programs and inflammatory responses .

What techniques can be employed to identify novel SETD6 interacting proteins using SETD6 antibodies?

Researchers can utilize SETD6 antibodies to discover novel interacting partners through these advanced methodological approaches:

• Immunoprecipitation Coupled with Mass Spectrometry:

  • Immunoprecipitate endogenous SETD6 from cells using specific antibodies conjugated to beads.

  • Use appropriate controls (beads only) to identify non-specific binding proteins.

  • Process samples for mass spectrometry analysis as described in Chen et al. :

    • Separate immunoprecipitated proteins by SDS-PAGE and stain with Coomassie.

    • Slice gels into 1mm squares for mass spectrometry analysis.

    • Analyze resulting peptides for protein identification.

• Proximity-Dependent Biotin Identification (BioID):

  • Generate fusion proteins of SETD6 with a promiscuous biotin ligase (BirA*).

  • Express in cells and add biotin to culture medium.

  • Proteins in close proximity to SETD6 become biotinylated and can be isolated with streptavidin.

  • Confirm candidate interactions using SETD6 antibodies in co-IP experiments.

• Yeast Two-Hybrid Screening with Validation:

  • Screen for SETD6 interactors using yeast two-hybrid system.

  • Validate positive hits in mammalian cells using SETD6 antibodies for co-IP confirmation.

  • Determine interaction domains through truncation mutants.

• In Vitro Binding Assays:

  • Express and purify recombinant SETD6 protein.

  • Incubate with cell lysates or protein fractions.

  • Use SETD6 antibodies to pull down complexes.

  • Identify binding partners through Western blotting or mass spectrometry.

• Network Analysis of Identified Interactors:

  • Use bioinformatics tools like STRING to analyze identified proteins .

  • Perform Gene Ontology (GO) enrichment analysis to identify biological processes.

  • Construct interaction networks as demonstrated in previous studies, which identified three major functional groups: metabolic processes, muscle contraction, and protein folding .

This systematic approach has already identified 115 new SETD6 cellular interacting protein candidates, providing valuable insights into SETD6's biological functions .

How can SETD6 antibodies be applied in cancer research, particularly for B-ALL studies?

SETD6 antibodies offer powerful tools for investigating SETD6's role in cancer development and progression, particularly in B-cell Acute Lymphoblastic Leukemia (B-ALL), through these advanced methodological approaches:

These methodologies enable researchers to thoroughly investigate SETD6's role in cancer development and progression, potentially identifying novel therapeutic targets and prognostic markers, particularly in B-ALL where SETD6 expression correlates with patient survival .

What are the key methodological considerations when using SETD6 antibodies for chromatin immunoprecipitation (ChIP) assays?

When utilizing SETD6 antibodies for ChIP assays to investigate SETD6's genomic targets and epigenetic functions, researchers should consider these critical methodological factors:

• Antibody Selection and Validation:

  • Choose SETD6 antibodies validated specifically for ChIP applications.

  • Test antibody specificity through Western blotting before ChIP experiments.

  • Validate ChIP efficiency using known SETD6 target regions (if available).

  • Consider using multiple antibodies targeting different SETD6 epitopes to confirm results .

• Cross-linking Optimization:

  • Optimize formaldehyde concentration (typically 1%) and cross-linking duration (10-15 minutes).

  • For studying transient interactions, consider using protein-protein cross-linkers in addition to formaldehyde.

  • Include glycine quenching (125 mM) to stop the cross-linking reaction effectively.

• Chromatin Preparation:

  • Optimize sonication conditions to achieve chromatin fragments of 200-500 bp.

  • Verify fragment size by agarose gel electrophoresis before proceeding.

  • Remove insoluble material by centrifugation (14,000 rpm for 10 minutes).

  • Pre-clear chromatin with protein A/G beads to reduce non-specific binding.

• Immunoprecipitation Protocol:

  • Use sufficient antibody amount (typically 2-5 μg per ChIP reaction).

  • Include appropriate controls:

    • IgG control (matching the host species of SETD6 antibody)

    • Input sample (10% of chromatin used for IP)

    • Positive control (antibody against abundant histone mark)

  • Allow sufficient incubation time (overnight at 4°C) for antibody binding.

  • Perform stringent washing steps to remove non-specific interactions.

• qPCR and Sequencing Considerations:

  • Design primers for regions where SETD6 is expected to bind based on literature.

  • Include control regions not expected to be bound by SETD6.

  • For ChIP-seq, ensure sufficient sequencing depth (typically >20 million reads).

  • Use appropriate peak calling algorithms sensitive enough for transcription factor binding.

• Data Analysis and Validation:

  • Normalize ChIP-qPCR data to input and IgG controls.

  • For ChIP-seq, perform replicate experiments and identify reproducible peaks.

  • Validate key findings with orthogonal techniques (e.g., reporter assays).

  • Correlate SETD6 binding with gene expression data to establish functional significance.

Following these methodological guidelines will optimize the detection of genuine SETD6 binding sites and provide valuable insights into SETD6's role in chromatin regulation and transcriptional control.

How can researchers utilize SETD6 antibodies to investigate the interplay between SETD6 and other epigenetic modifiers?

SETD6 antibodies enable sophisticated investigations into the functional relationships between SETD6 and other epigenetic regulators through these advanced methodological approaches:

• Sequential ChIP (Re-ChIP) Analysis:

  • Perform initial ChIP with SETD6 antibodies.

  • Elute the protein-DNA complexes.

  • Perform a second ChIP with antibodies against other epigenetic modifiers.

  • This technique reveals genomic loci where SETD6 co-occupies with other epigenetic factors.

• Co-Immunoprecipitation of Epigenetic Complexes:

  • Use SETD6 antibodies to immunoprecipitate native protein complexes.

  • Analyze by Western blot for known epigenetic modifiers (e.g., HDACs, DNMTs, other HMTs).

  • Alternatively, perform mass spectrometry analysis to identify all associated proteins .

  • Previous studies have identified 115 SETD6 interacting proteins, many involved in epigenetic processes .

• Proximity Ligation Assay (PLA):

  • Utilize SETD6 antibodies alongside antibodies against candidate interacting epigenetic modifiers.

  • Quantify in situ protein-protein interactions at single-molecule resolution.

  • Assess how these interactions change under different cellular conditions or treatments.

• Combinatorial ChIP-seq Analysis:

  • Perform parallel ChIP-seq experiments with SETD6 antibodies and antibodies against histone modifications or other epigenetic factors.

  • Identify regions of overlap and mutual exclusion.

  • Correlate binding patterns with gene expression data to determine functional significance.

• Epigenetic Inhibitor Studies:

  • Treat cells with specific inhibitors of various epigenetic pathways.

  • Use SETD6 antibodies to assess changes in SETD6 localization, binding partners, or activity.

  • For example, monitor changes in SETD6 expression after treatment with DNA methyltransferase inhibitors like AZA, which has been shown to restore SETD6 expression in hypermethylated cell lines .

This multi-faceted approach provides a comprehensive understanding of how SETD6 functions within the broader epigenetic regulatory network, revealing potential crosstalk mechanisms and co-regulatory relationships that may be targeted for therapeutic intervention in diseases like cancer.

What protocols should be followed when using SETD6 antibodies for super-resolution microscopy?

When employing SETD6 antibodies for super-resolution microscopy techniques such as STORM, PALM, or STED, researchers should implement these specialized methodological considerations:

• Antibody Selection and Modification:

  • Choose high-affinity SETD6 antibodies with proven specificity in immunofluorescence applications .

  • For direct STORM, conjugate antibodies to photoswitchable fluorophores (e.g., Alexa Fluor 647).

  • For PALM, consider using SETD6 fusion proteins with photoactivatable fluorescent proteins instead of antibodies.

  • For STED, select antibodies conjugated with photostable dyes (e.g., ATTO or Star dyes).

• Sample Preparation Optimization:

  • Fix cells with 4% paraformaldehyde (10 minutes at room temperature).

  • For cytoplasmic proteins, permeabilize with 0.1-0.2% Triton X-100.

  • For nuclear proteins like SETD6, use 0.5% Triton X-100 for better nuclear penetration.

  • Consider alternative fixation methods (e.g., methanol) if standard protocols yield high background.

  • Implement additional blocking steps (2-3 hours) with 5% BSA and 5% normal serum to minimize non-specific binding.

• Antibody Incubation Parameters:

  • Dilute primary SETD6 antibodies appropriately (typically 1:100 to 1:500).

  • Extend primary antibody incubation to overnight at 4°C to maximize specific binding.

  • For direct detection, use fluorophore-conjugated SETD6 antibodies.

  • For indirect detection, select secondary antibodies with brightness and photostability suited for super-resolution imaging.

  • Include extensive washing steps (5-6 washes of 5 minutes each) to remove unbound antibodies.

• Multicolor Imaging Considerations:

  • When co-imaging SETD6 with other proteins of interest:

    • Select fluorophores with minimal spectral overlap.

    • Perform sequential imaging to prevent crosstalk.

    • Include controls for each fluorophore individually to confirm specificity.

• Imaging Buffer Optimization:

  • For STORM: Use oxygen-scavenging system (glucose oxidase/catalase) with thiol compound (MEA).

  • For STED: Use ProLong Glass or TDE-based mounting media to match refractive index.

  • For all methods: Filter all buffers and mounting media (0.22 μm filter) to remove fluorescent particles.

• Validation and Quantification:

  • Include appropriate controls (secondary-only, IgG controls, SETD6 knockdown cells).

  • Perform conventional microscopy in parallel for comparison.

  • Use clustering algorithms to analyze SETD6 distribution patterns.

  • Quantify co-localization with other proteins at nanoscale resolution.

These optimized protocols enable researchers to visualize SETD6 localization and interactions at unprecedented resolution, revealing detailed insights into its spatial organization and functional compartmentalization within cellular structures.

How should researchers choose between different epitope-specific SETD6 antibodies for particular applications?

Selecting the optimal epitope-specific SETD6 antibody requires careful evaluation of multiple factors based on the intended application. This methodological decision-making framework will guide researchers in making informed choices:

• Epitope Location Considerations:

  Epitope Region	Advantages	Recommended Applications
  N-terminal (AA 1-74)	- Often more accessible in native protein - Suitable for detecting full-length SETD6 - Less affected by post-translational modifications	- Western blotting - ELISA - IHC of fixed tissues
  Central region	- Contains functional domains - May be blocked in protein complexes	- Detection of free SETD6 - Studies of SETD6 domain function
  C-terminal (AA 310-338, 359-408)	- Often exposed on protein surface - May detect truncated forms - Useful when N-terminus is processed or blocked	- Detecting various SETD6 isoforms - C-terminal processing studies

• Application-Specific Selection Criteria:

  • For Western Blotting: Choose antibodies validated specifically for WB with demonstrated ability to detect denatured SETD6 .

  • For Immunoprecipitation: Select antibodies recognizing native epitopes, preferably with demonstrated IP efficiency in published studies .

  • For IHC/ICC: Choose antibodies validated for these applications with minimal background staining in control samples .

  • For ChIP Applications: Select antibodies recognizing epitopes not involved in DNA binding to avoid interference with chromatin interactions.

• Experimental Validation Strategy:

  • Test multiple SETD6 antibodies targeting different epitopes in parallel.

  • Compare performance using positive controls (cells with known SETD6 expression) and negative controls (SETD6 knockdown).

  • For critical studies, validate key findings with at least two antibodies targeting different SETD6 epitopes.

• Special Considerations for HRP-Conjugated Antibodies:

  • Direct HRP conjugation may reduce antibody affinity in some cases.

  • The large HRP molecule might cause steric hindrance for certain epitopes.

  • HRP-conjugated antibodies are ideal for applications requiring direct detection but may be less versatile than unconjugated versions.

  • Consider epitope accessibility when using HRP-conjugated antibodies for detection of native SETD6 in complex samples.

• Decision Matrix for Selecting Optimal SETD6 Antibody:

  Research Question	Recommended Epitope	Conjugation	Clonality
  SETD6 expression levels	N-terminal (AA 1-74)	HRP for direct detection	Polyclonal for broader epitope recognition
  SETD6 protein interactions	Multiple epitopes to avoid interference with binding sites	Unconjugated for IP applications	Both mono and polyclonal for validation
  SETD6 in cancer studies	Epitopes shown to be preserved in cancer tissues	Application-dependent	Monoclonal for consistency across samples
  SETD6 localization	Accessible epitopes in fixed cells	Fluorescent or HRP	Validated for specific microscopy application

By systematically evaluating these factors, researchers can select the most appropriate SETD6 antibody for their specific experimental requirements, ensuring optimal results and reliable data interpretation.

How can researchers implement integrative approaches using SETD6 antibodies to understand its role in disease progression?

To comprehensively investigate SETD6's role in disease, particularly in cancer progression, researchers should implement an integrative, multi-technique approach utilizing SETD6 antibodies:

This integrated approach enables researchers to establish comprehensive connections between SETD6's molecular functions and its roles in disease pathogenesis, potentially identifying novel therapeutic targets and prognostic indicators.