SETD5 Antibody

What is SETD5 and what is its molecular function?

SETD5 (SET domain containing 5) is a member of the histone lysine methyltransferase family and is involved in regulating gene expression through modifications in chromatin structure. Despite its annotation as a candidate lysine methyltransferase that potentially methylates histone H3 on lysine 36 (H3K36), recent evidence suggests that SETD5 may actually lack intrinsic methyltransferase activity. Instead, it appears to function as a scaffold for a co-repressor complex that includes HDAC3 and G9a, which couples methylation of H3K9 with deacetylation of this residue .

SETD5 has been implicated in various cellular functions including:

• Transcriptional regulation

• Euchromatin formation

• RNA elongation and splicing

• Early embryonic development

• Cell cycle regulation and proliferation

Additionally, SETD5 has gained significant attention for its role in neurodevelopmental disorders, particularly Autism Spectrum Disorder (ASD), and its involvement in cancer therapy resistance mechanisms .

What are the typical molecular weight bands observed for SETD5 in western blotting?

The molecular weight of SETD5 protein reported in literature shows some variation:

This discrepancy highlights a critical issue in SETD5 research. Newman et al. reported that the ThermoFisher SETD5 antibody (PA-53718) showed multiple bands, including one at the predicted size of 150 kDa and another at 60 kDa that ThermoFisher had defined as a correct SETD5 band. This inconsistency led to significant confusion, ultimately resulting in ThermoFisher removing the antibody from their catalog .

When working with SETD5 antibodies, researchers should anticipate potential bands in the 150-220 kDa range for full-length protein, while being aware that shorter isoforms or degradation products might be detected at lower molecular weights.

How can researchers verify the specificity of SETD5 antibodies?

Given the documented issues with antibody specificity for SETD5, rigorous validation is essential. The following methodological approaches are recommended:

• Genetic controls: Use SETD5 knockout or knockdown systems as negative controls. Newman et al. demonstrated that analyzing samples from SETD5 knockout embryos provides definitive evidence of antibody specificity .

• Recombinant protein testing: Test antibody against purified recombinant SETD5 protein of known concentration to establish detection limits and specificity.

• Multiple antibody comparison: Use at least two antibodies targeting different epitopes of SETD5 to cross-validate results.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide to confirm that binding is specific to the target epitope.

• Mass spectrometry validation: For definitive identification, consider immunoprecipitation followed by mass spectrometry to confirm the identity of the detected proteins.

The experience reported by Newman et al. highlights why these validation steps are critical - their investigation of the ThermoFisher SETD5 antibody revealed inconsistencies that ultimately led to the removal of the product from the market .

What are the known structural and functional domains of SETD5 relevant to antibody selection?

Understanding SETD5's domain architecture is crucial for rational antibody selection:

SETD5 contains:

• A SET domain (the defining feature of this protein family)

• Several conserved regions that differentiate it from other SET domain proteins

The domain organization of human SETD5 (in comparison to related proteins) includes:

When selecting antibodies, researchers should consider:

• Which domain the antibody targets (N-terminal, C-terminal, or SET domain)

• Whether the epitope is accessible in experimental conditions

• If the targeted region is conserved across species (if cross-reactivity is desired)

How can researchers optimize western blotting protocols specifically for SETD5 detection?

Optimizing western blotting for SETD5 detection requires special consideration due to its high molecular weight and reported detection issues:

• Sample preparation optimization:

  • Newman et al. identified specific lysis buffer compositions and sonication conditions that improved signal-to-noise ratio

  • Consider using single-stranded binding protein (SSB) to improve antibody specificity

• Gel electrophoresis modifications:

  • Use lower percentage gels (6-8%) to better resolve the high molecular weight SETD5 (160-220 kDa)

  • Extend running time to improve separation of high molecular weight proteins

• Transfer optimization:

  • Use wet transfer methods with extended transfer times for high molecular weight proteins

  • Consider adding SDS (0.1%) to transfer buffer to facilitate movement of large proteins

• Blocking and antibody incubation:

  • Optimal dilutions reported for western blotting range from 1:500 to 1:2000

  • Extended primary antibody incubation (overnight at 4°C) may improve sensitivity

• Detection considerations:

  • For weaker signals, consider using enhanced chemiluminescence or fluorescence-based detection systems

  • Longer exposure times may be necessary for detecting endogenous SETD5 in some cell types

Newman et al. reported that direct PCR amplification of membrane pieces provided optimal conditions for generating sequencing libraries in their western-seq system, which could be adapted for traditional western blotting applications .

What is the role of SETD5 in neurodevelopmental disorders and how can antibodies help investigate this function?

SETD5 has been implicated in Autism Spectrum Disorder (ASD) and other neurodevelopmental disorders, though there is disagreement in the literature about its precise molecular role .

Researchers investigating SETD5's role in neurodevelopmental disorders can utilize antibodies in the following methodological approaches:

• Tissue expression profiling:

  • Immunohistochemistry (IHC) to map SETD5 expression patterns in neural tissues during development

  • Compare expression in normal vs. pathological tissues from model organisms or post-mortem samples

• Protein complex identification:

  • Co-immunoprecipitation with SETD5 antibodies followed by mass spectrometry to identify interaction partners in neural cells

  • This approach can reveal how SETD5 functions in neurodevelopmental pathways

• Chromatin association studies:

  • Chromatin immunoprecipitation (ChIP) using SETD5 antibodies to identify genomic regions bound by SETD5

  • Integration with transcriptomic data to identify genes regulated by SETD5 in neural development

• Post-translational modification analysis:

  • Immunoprecipitation with SETD5 antibodies followed by mass spectrometry to identify modifications that may regulate its function in neural cells

Recent studies suggest that SETD5 may function as a scaffold for co-repressor complexes rather than as an active methyltransferase, which has implications for understanding its role in neurodevelopment .

How does SETD5 contribute to cancer therapy resistance and what experimental approaches can investigate this mechanism?

Recent research has identified SETD5 as a major driver of resistance to MEK1/2 inhibition (MEKi) therapy in pancreatic ductal adenocarcinoma (PDAC) . This finding opens new avenues for investigation using SETD5 antibodies:

• Resistance mechanism characterization:

  • SETD5 coordinates chromatin reprogramming that regulates adaptive resistance to targeted therapy

  • SETD5 scaffolds a co-repressor complex including HDAC3 and G9a to silence genes involved in drug sensitivity

• Experimental approach using antibodies:

  • Western blotting to monitor SETD5 expression changes during development of resistance

  • Chromatin immunoprecipitation (ChIP) to identify changing patterns of SETD5 genomic binding during therapy

  • Co-immunoprecipitation to confirm interaction with HDAC3 and G9a in resistant cells

• Therapeutic implications:

  • Deletion of SETD5 restored vulnerability to MEKi therapy in both mouse models and patient-derived xenografts

  • Pharmacological co-targeting of MEK1/2, HDAC3, and G9a sustained tumor growth inhibition in vivo

• Monitoring therapy response:

  • Immunohistochemistry with SETD5 antibodies could potentially serve as a biomarker for predicting response to MEK inhibitors

  • Observed overexpression of SETD5 in pancreatic cancer suggests potential diagnostic applications

This research demonstrates how SETD5 antibodies can be instrumental in elucidating complex epigenetic mechanisms of therapy resistance and identifying new therapeutic targets.

What are the optimal storage and handling conditions for SETD5 antibodies?

Based on the commercial antibody information provided, the following storage and handling recommendations apply to SETD5 antibodies:

Proper storage and handling are essential for maintaining antibody performance and reproducibility across experiments .

How can researchers address multiple band detection and specificity issues with SETD5 antibodies?

Multiple band detection has been a documented issue with SETD5 antibodies, as highlighted by Newman et al. . To address this challenge:

• Detailed characterization of all detected bands:

  • Document the precise molecular weights of all detected bands

  • Compare with predicted molecular weights of known SETD5 isoforms (SETD5 has at least 3 reported isoforms )

• Validation with genetic approaches:

  • SETD5 knockdown or knockout to confirm which bands are specific

  • Newman et al. demonstrated that SETD5 knockout did not affect ROSA26 expression, but disruption of ROSA26 did affect SETD5 expression, revealing complex regulatory relationships

• Advanced protein analysis:

  • Mass spectrometry identification of immunoprecipitated proteins to confirm band identity

  • Newman et al. developed a novel protein quantification method called "western-seq" that merges qualitative size information with quantitative DNA sequencing data using antibodies conjugated to oligonucleotides

• Consider alternative detection methods:

  • If western blotting proves problematic, consider alternative applications like immunofluorescence or ELISA

  • Combination of methods provides stronger validation of results

The inconsistency reported with the ThermoFisher SETD5 antibody (showing bands at both 150 kDa and 60 kDa) demonstrates why these validation steps are critical for meaningful research outcomes .

What species cross-reactivity can be expected with currently available SETD5 antibodies?

The species reactivity of commercially available SETD5 antibodies varies by product:

When studying SETD5 in model organisms, researchers should:

• Verify the sequence homology between the target epitope in the model organism and human SETD5

• Perform validation experiments in the specific organism of interest

• Consider using an antibody specifically raised against the species-specific SETD5 sequence when available

The conservation of SETD5 across species makes it possible to use some antibodies across multiple organisms, but validation is essential for each new species application .

How can SETD5 antibodies contribute to understanding the protein's role in epigenetic regulation?

SETD5 appears to participate in epigenetic regulation through a scaffolding role rather than direct histone methyltransferase activity. Antibodies can help elucidate this function through:

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • Mapping SETD5 binding sites across the genome

  • Integration with histone modification data (H3K9me, H3K36me) to understand functional relationships

• Protein complex analysis:

  • Co-immunoprecipitation with SETD5 antibodies to identify and confirm interaction partners

  • Evidence suggests SETD5 scaffolds a co-repressor complex including HDAC3 and G9a

• Functional domain analysis:

  • Using antibodies targeting different domains of SETD5 to understand domain-specific functions

  • Comparison with other SET domain family members like MLL5, Set3, Set4, and UpSET

• Combinatorial epigenetic profiling:

  • Simultaneous profiling of SETD5 binding and histone modifications

  • Integration with transcriptomic data to link epigenetic changes to gene expression outcomes

These approaches can help resolve the current disagreement in literature about SETD5's molecular role and provide insight into how mutations in this gene contribute to neurodevelopmental disorders and cancer .

What novel methodologies are being developed to improve SETD5 protein detection and quantification?

Newman et al. described the development of an innovative approach called "western-seq" that addresses limitations in current antibody-based protein quantification methods:

• Western-seq methodology:

  • Merges qualitative size information from western blotting with quantitative DNA sequencing data

  • Uses antibodies conjugated to oligonucleotides for detection

  • Demonstrated improved signal-to-noise ratio under optimized conditions

• Optimized conditions identified:

  • Specific lysis buffer compositions

  • Defined sonication conditions

  • Addition of single-stranded binding protein (SSB)

  • Direct PCR amplification of membrane pieces for generating sequencing libraries

• Validation approach:

  • Initial testing with conjugated antibodies and recombinant protein complexes

  • Subsequent validation in HEK293 cells

  • Comparison with traditional western blotting methods

This methodological innovation demonstrates how challenges with SETD5 antibody specificity have driven the development of novel approaches that may have broader applications across protein detection techniques.

How can SETD5 antibodies be applied in studying therapeutic approaches for SETD5-associated disorders?

SETD5 antibodies can play crucial roles in developing and evaluating therapeutic approaches for both neurodevelopmental disorders and cancer:

• Therapeutic target validation:

  • Western blotting and immunohistochemistry to confirm SETD5 expression in disease models

  • Monitoring changes in SETD5 expression or localization in response to therapeutic interventions

• Drug development applications:

  • High-throughput screening assays using SETD5 antibodies to identify compounds that modulate its function or expression

  • Evaluation of drugs targeting the SETD5 co-repressor complex (HDAC3, G9a)

• Combination therapy approach:

  • Wang et al. demonstrated that pharmacological co-targeting of MEK1/2, HDAC3, and G9a sustained tumor growth inhibition in pancreatic cancer

  • SETD5 antibodies can monitor changes in complex formation during treatment

• Biomarker development:

  • Immunohistochemistry with SETD5 antibodies to stratify patients for clinical trials

  • Potential diagnostic applications given SETD5's overexpression in certain cancers