setd6 Antibody

What is SETD6 and why is it important in research?

SETD6 (SET domain containing 6) is a protein lysine methyltransferase with significant roles in cellular processes including transcriptional regulation and stem cell differentiation. In humans, the canonical SETD6 protein consists of 473 amino acid residues with a molecular mass of 53.2 kDa and is primarily localized in the nucleus . SETD6 contains a SET domain and belongs to the class V-like SAM-binding methyltransferase superfamily . It has gained importance in research due to its ability to methylate various protein substrates, including BRD4, TWIST1, and the RELA subunit of NF-κB, which affects multiple biological processes from gene expression to learning and memory formation .

What types of SETD6 antibodies are available for research applications?

SETD6 antibodies are available in various formats including:

• Monoclonal antibodies (e.g., PCRP-SETD6-1E8)

• Polyclonal antibodies from different host species (primarily rabbit)

• Antibodies targeting different epitopes/regions of SETD6 (N-terminal, middle region, C-terminal)

• Conjugated antibodies (FITC, biotin, HRP) for specialized applications

• Site-specific antibodies that recognize methylated SETD6 target proteins

How should I select the appropriate SETD6 antibody for my research?

Selection criteria should include:

Review literature in your specific research area to identify which SETD6 antibodies have been successfully used for similar experiments .

What are the optimal conditions for using SETD6 antibodies in Western blotting?

For optimal Western blot results with SETD6 antibodies:

• Sample preparation:

  • Extract nuclear proteins as SETD6 is primarily located in the nucleus

  • Include protease inhibitors to prevent degradation

  • For chromatin-associated SETD6, consider chromatin fractionation methods

• Recommended dilutions:

  • Typical working dilutions range from 1:500 to 1:2000

  • Optimal dilution is antibody-specific; for example, Proteintech's 24377-1-AP antibody works best at 1:500-1:1000

• Detection specifics:

  • Expected molecular weight: 53 kDa

  • Consider using positive control lysates (e.g., RAW 264.7 cells have been verified for some antibodies)

  • For SETD6 knockout validation, CRISPR/Cas9-generated SETD6 KO cells can serve as negative controls

• Validation approach:

  • When studying SETD6-mediated methylation, include methylation-deficient mutants (e.g., K33R for TWIST1 studies)

  • Knockdown validation using siRNA against SETD6 can confirm antibody specificity

How can I optimize immunoprecipitation experiments with SETD6 antibodies?

For successful SETD6 immunoprecipitation:

• Lysis conditions:

  • Use nuclear extraction buffers containing 150-300 mM NaCl

  • Include methylation-preserving inhibitors if studying methylated targets

  • Consider crosslinking for transient interactions

• IP approach:

  • For detecting SETD6 interactions, immunoprecipitation of tagged SETD6 (FLAG-SETD6) has been successfully used to pull down interacting partners including MTA2, TAF4, and TRRAP

  • Commercial SETD6 antibodies like PCRP-SETD6-1E8 have been validated for immunoprecipitation applications

• Validation strategies:

  • Verify results with reciprocal IPs (e.g., IP SETD6 and blot for partner, then IP partner and blot for SETD6)

  • For studying SETD6-dependent methylation, compare results between wild-type and SETD6 knockout cells

• Complex detection:

  • When studying SETD6-containing complexes, consider using antibodies to known interactors (e.g., HDAC1, MTA2)

  • For studying methylated targets of SETD6, develop or obtain site-specific methyl-lysine antibodies

What are the best methods to study SETD6-mediated protein methylation?

Investigating SETD6-mediated methylation requires multiple approaches:

• In vitro methylation assays:

  • Use recombinant SETD6 and purified substrate proteins

  • Include S-adenosylmethionine (SAM) as methyl donor

  • Analyze by autoradiography ([³H]-SAM) or Western blotting with methyl-specific antibodies

• Mass spectrometry analysis:

  • Perform non-radioactive methylation assays followed by mass spectrometry to map specific methylation sites

  • Confirm with site-directed mutagenesis of candidate lysine residues to arginine (e.g., K33R for TWIST1)

• Site-specific methyl-lysine antibodies:

  • Generate antibodies against methylated peptides corresponding to SETD6 target sites

  • Validate with peptide competition assays and methylation-deficient mutants

  • Examples include antibodies against BRD4-K99me1 and TWIST1-K33me1

• Cell-based validation:

  • Compare methylation in wild-type versus SETD6 knockout cells

  • Rescue experiments with wild-type SETD6 but not catalytically inactive mutants

  • Analyze phenotypic consequences of mutations at methylation sites

How can I investigate SETD6's role in transcriptional regulation using antibodies?

SETD6 plays complex roles in transcriptional regulation that can be studied using antibodies in several advanced approaches:

• Chromatin immunoprecipitation (ChIP):

  • Use SETD6 antibodies to identify genomic binding sites

  • For HPV research, SETD6 has been found to localize specifically to the enhancer region of the long control region (LCR)

  • Combine with antibodies against histone marks (e.g., H3K27me3) to correlate SETD6 binding with chromatin state

• Sequential ChIP (re-ChIP):

  • Perform to determine co-occupancy of SETD6 with interacting partners

  • Study has shown SETD6 co-occupancy with BRD4 at viral enhancer regions

• Transcriptional reporter assays:

  • Compare effects of wild-type versus methylation-deficient mutants

  • SETD6 has been shown to affect estrogen-responsive gene expression and GAL4-responsive reporters

• Complex analyses:

  • Use co-immunoprecipitation with SETD6 antibodies to detect interactions with transcriptional regulators (e.g., HDAC1, MTA2, ERα)

  • SETD6 associates with both corepressors (HDAC1) and coactivators (TRRAP)

What approaches can I use to study SETD6's involvement in mRNA translation regulation?

Recent research has revealed SETD6's role in regulating mRNA translation through BRD4 methylation :

• Puromycin incorporation assay:

  • Measure global protein synthesis rates in SETD6 knockout versus control cells

  • Puromycin is incorporated into newly synthesized peptides and can be detected by Western blot with anti-puromycin antibodies

  • Research has shown elevated protein synthesis in SETD6 KO cells

• Polysome profiling:

  • Analyze monosome and polysome fractions to detect changes in translation efficiency

  • Western blot analysis of fractions can determine if SETD6 or its targets associate with translation machinery

  • Studies showed BRD4 was not directly associated with monosome or polysome fractions

• BRD4 methylation assessment:

  • Use site-specific antibodies against BRD4-K99me1 to correlate methylation status with translation rates

  • Compare wild-type BRD4 with K99R mutant effects on translation

• RNA-seq and ribosome profiling:

  • Identify translation-related genes regulated by SETD6

  • Correlate with BRD4 occupancy at these gene promoters

How can I investigate SETD6's role in cellular processes like cell migration and adhesion?

SETD6 regulates cell migration and adhesion through TWIST1 methylation and LINC-PINT expression :

• Migration and adhesion assays:

  • Compare wild-type cells to SETD6 knockout cells

  • Rescue experiments with wild-type SETD6 versus methylation-deficient mutants

• TWIST1 methylation analysis:

  • Use site-specific antibodies against TWIST1-K33me1

  • Compare effects of wild-type TWIST1 versus K33R mutant on cell phenotypes

• ChIP analysis of TWIST1 targets:

  • Study TWIST1 and EZH2 occupancy at the LINC-PINT locus

  • Correlate with H3K27me3 levels and LINC-PINT expression

• RNA expression analysis:

  • Measure LINC-PINT expression levels in relation to SETD6 activity

  • Correlate with cellular phenotypes (adhesion, migration)

How can I validate the specificity of SETD6 antibody signals in my experiments?

Multiple validation approaches should be employed:

• Genetic validation:

  • Use SETD6 knockout cells generated via CRISPR/Cas9 as negative controls

  • siRNA knockdown of SETD6 to demonstrate signal reduction

  • Rescue experiments with exogenous SETD6 expression in knockout cells

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide to block specific binding

  • Include scrambled peptides as controls

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of SETD6

  • Compare results from monoclonal and polyclonal antibodies

• Application-specific controls:

  • For ChIP experiments, include IgG controls and compare enrichment to known target regions

  • For site-specific methylation antibodies, use methylation-deficient mutants (e.g., K33R for TWIST1)

What are common pitfalls when working with SETD6 antibodies and how can I address them?

Common challenges and solutions include:

• Nuclear localization challenges:

  • Ensure proper nuclear extraction protocols as SETD6 is primarily nuclear

  • For immunofluorescence, optimize fixation conditions to preserve nuclear structure

• Cross-reactivity issues:

  • Site-specific methyl-lysine antibodies may show cross-reactivity with unmethylated proteins

  • Control for this by including in vitro methylation reactions with and without SETD6

• Signal intensity problems:

  • SETD6 expression may be low in some cell types

  • Consider immunoprecipitation before Western blotting to concentrate the protein

• Conflicting results:

  • SETD6 can function as both an activator and repressor depending on context

  • Carefully consider cell type and experimental conditions when interpreting results

  • When studying methylation targets, consider that other methyltransferases might compensate in SETD6 knockout systems

How can I differentiate between SETD6-mediated effects and those of other SET domain proteins?

Distinguishing SETD6-specific effects requires:

• Substrate specificity analysis:

  • Compare methylation patterns of known SETD6 targets (BRD4-K99, TWIST1-K33, RELA-K310) versus targets of other SET proteins

  • Use site-specific methyl-lysine antibodies to monitor specific methylation events

• Rescue experiments:

  • In SETD6 knockout models, perform rescue with wild-type SETD6 versus:

    • Catalytically inactive SETD6 mutants

    • Other SET domain proteins

  • Analyze whether the phenotype is specifically restored by SETD6

• Protein interaction networks:

  • Compare SETD6-associated proteins (HDAC1, MTA2, TRRAP) with interaction partners of other SET proteins

  • Use this information to predict SETD6-specific pathways

• Bioinformatic approaches:

  • Analyze substrate specificity based on amino acid context of methylation sites

  • SETD6 has been shown to have preference for glycine-lysine motifs

How can SETD6 antibodies be used to investigate its role in learning and memory formation?

SETD6 plays a critical role in learning-induced processes in the hippocampus :

• In vivo approaches:

  • Use site-specific delivery of siRNA against SETD6 in rodent models

  • Monitor changes in learning-associated RELA-K310 methylation and H3K9me2 using specific antibodies

• Electrophysiological correlations:

  • Combine with electrophysiological recordings to correlate SETD6 activity with synaptic plasticity

  • Analyze NF-κB transcription factor binding using electrophoretic mobility shift assays (EMSA)

• Behavioral paradigms:

  • Use contextual fear conditioning (CFC) to induce learning

  • Analyze SETD6-dependent molecular changes at different time points after learning

• Region-specific analyses:

  • Monitor SETD6 expression and activity in specific brain regions (e.g., CA1 of hippocampus)

  • Compare with other brain regions to determine localization of effects

What new methodologies are being developed for studying SETD6 function in complex biological systems?

Emerging approaches include:

• CRISPR-based techniques:

  • CRISPR activation/interference to modulate SETD6 expression without genetic knockout

  • CRISPR base editing to introduce specific mutations in SETD6 or its targets (e.g., K33R in TWIST1)

• Proximity labeling:

  • BioID or APEX2 fusions with SETD6 to identify proximal proteins in living cells

  • Could reveal transient interactions missed by traditional immunoprecipitation

• Single-cell analyses:

  • Single-cell RNA-seq to identify cell populations dependent on SETD6 function

  • Correlation with single-cell proteomics for comprehensive understanding

• Advanced imaging:

  • Super-resolution microscopy to visualize SETD6 localization at specific genomic loci

  • Live-cell imaging of SETD6 dynamics during cellular processes

How might SETD6 antibodies contribute to understanding disease mechanisms and therapeutic development?

SETD6 has emerging roles in various diseases:

• Cancer research applications:

  • SETD6 regulates BRD4 and TWIST1, both implicated in cancer progression

  • Antibodies can monitor SETD6 expression and activity in tumor samples

• Viral infection studies:

  • SETD6 regulates HPV transcription through BRD4 methylation

  • Antibodies can track SETD6 recruitment to viral genomic elements

• Neurological disorders:

  • Given SETD6's role in learning and memory, antibodies can assess its function in neurological disease models

  • Monitor RELA-K310 methylation as a readout of SETD6 activity in brain tissue

• Drug development:

  • Screen for compounds that modulate SETD6 activity using antibody-based assays

  • Monitor on-target effects of SETD6 inhibitors using specific antibodies against SETD6 and its methylated targets