setd7 Antibody

What is SETD7 and why is it significant in molecular research?

SETD7 exhibits notably broad target specificity, methylating various non-histone proteins including transcriptional regulators such as:

• p53/TP53 (stabilizing p53 and increasing transcriptional activation)

• TAF10 (increasing its affinity for RNA polymerase II)

• TAF7, ER, p65, STAT3, Rb, Mypt, Tat, and Foxo3

The enzyme plays central roles in transcriptional activation of genes such as collagenase and insulin . Interestingly, recent research has revealed context-dependent functions, acting as both a transcriptional activator and repressor depending on cellular context .

What are the key differences between monoclonal and polyclonal SETD7 antibodies?

When selecting between these antibody types, consider your experimental goals. Monoclonal antibodies offer superior specificity and consistency but may be less sensitive for detecting proteins with low expression levels. Polyclonal antibodies provide higher sensitivity due to multiple epitope recognition but may show batch-to-batch variation .

How does SETD7 localization vary across different cell types and experimental conditions?

Contrary to assumptions about nuclear localization for a histone-modifying enzyme, studies show that SETD7 displays predominantly cytoplasmic localization in various cell types:

For accurate localization studies:

• Use both immunofluorescence and subcellular fractionation followed by western blotting

• Include appropriate compartment markers (e.g., GAPDH for cytoplasm, Lamin B for nucleus)

• Consider cross-validation with different SETD7 antibodies to ensure epitope accessibility isn't affecting localization detection

What are the optimal protocols for using SETD7 antibodies in different experimental applications?

Western Blot (WB) Protocol:

• Recommended dilution: 1:1000-1:5000 for polyclonal antibodies ; 1:500-1:2000 for monoclonal antibodies

• Positive samples: HeLa cells, C6, mouse brain tissue, mouse kidney tissue, NIH/3T3 cells, rat brain tissue

• Expected molecular weight: Calculated 41 kDa; Observed 48-50 kDa (likely due to post-translational modifications)

• MCF-7 nuclear extract can serve as a positive control

Immunofluorescence (IF)/ICC Protocol:

• Recommended dilution: 1:20-1:200 for polyclonal antibodies ; 1:100 for monoclonal antibodies

• Positive samples: HeLa cells have been validated

• Cellular localization varies by cell type (see FAQ #3)

Co-immunoprecipitation (Co-IP) Protocol:

• Harvest cells and wash three times in cold PBS

• Lyse in buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, pH 8.0, with protease inhibitors)

• Incubate lysates with SETD7 antibody overnight at 4°C with gentle shaking

• Add magnetic beads for 2-4 hours

• Wash beads three times with PBS

• Resuspend in 2× loading buffer and boil for 10 minutes

• Collect liquid for immunoblotting analysis

How can SETD7 antibodies be utilized in cancer research to investigate its context-dependent roles?

SETD7 exhibits diverse, sometimes contradictory roles in different cancer types:

Clear Cell Renal Cell Carcinoma (ccRCC):

• SETD7 is significantly upregulated in ccRCC tissues compared to adjacent normal tissues

• Knockdown of SETD7 in ccRCC cell lines (786-O and CAKI-1) inhibits cell proliferation, reduces colony formation, affects cell cycle (decreases G1 phase cells, increases S phase cells), and promotes apoptosis

• SETD7 silencing suppresses ccRCC cell metastasis

Prostate Cancer:

• SETD7 functions as a transcriptional repressor in castration-resistant prostate cancer (CRPC) cells

• SETD7-repressed genes are dramatically increased in CRPC compared to normal and primary prostate cancer

• SETD7 binding is markedly associated with its repression function rather than canonical activation function

Methodological approaches:

• Expression analysis: Compare SETD7 levels between tumor and normal tissues using IHC, qRT-PCR, and western blotting

• Functional studies: Use siRNA knockdown or CRISPR-Cas9 to examine effects on proliferation, migration, and apoptosis

• Mechanistic investigations: Combine SETD7 immunoprecipitation with mass spectrometry to identify cancer-specific interacting partners

• Chromatin binding analysis: Use ChIP-seq with SETD7 antibodies to map context-specific binding sites

How can researchers investigate SETD7-mediated methylation of non-histone proteins?

SETD7 methylates numerous non-histone proteins, affecting their stability, activity, and interactions. To investigate these modifications:

Co-immunoprecipitation coupled with methylation detection:

• Perform Co-IP with SETD7 antibody to pull down SETD7 and associated proteins

• Analyze by western blot using antibodies against suspected target proteins

• Use pan-methyl-lysine antibodies to detect methylation status

Example with LC3B in ovarian cancer:

• Endogenous SETD7 interacts with endogenous LC3B (demonstrated by immunoprecipitation)

• SETD7 overexpression reduces LC3B protein levels without affecting mRNA expression

• SETD7 promotes ubiquitination of LC3B, targeting it for degradation by the ubiquitin-proteasome system

Example with TAF7 in ccRCC:

• Protein docking analysis predicted binding between SETD7 and TAF7

• Co-immunoprecipitation confirmed interaction between these proteins

• SETD7-mediated TAF7 methylation regulates transcriptional activation of CCNA2

For validating methylation events:

• Generate methylation-specific antibodies for your protein of interest

• Use mass spectrometry to identify specific methylated residues

• Create methylation-deficient mutants to confirm functional consequences

Why might SETD7 appear at a different molecular weight than predicted, and how should researchers address this?

SETD7 has a calculated molecular weight of 41 kDa (366 amino acids), but is typically observed at 48-50 kDa in western blots . This discrepancy is important to understand when validating antibodies and interpreting results.

Possible explanations:

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications can increase apparent molecular weight

• Isoforms: Alternative splicing may generate different isoforms with varying molecular weights

• Structural properties: Some proteins migrate anomalously on SDS-PAGE due to unusual amino acid composition or conformational properties

Recommendations for addressing molecular weight discrepancies:

• Validate with positive controls: Use samples known to express SETD7 (e.g., HeLa cells, mouse brain tissue)

• Include knockdown/knockout controls: Compare with samples where SETD7 is silenced via siRNA or CRISPR-Cas9

• Cross-validate with multiple antibodies: Test different antibodies targeting distinct epitopes of SETD7

• Perform immunoprecipitation: Followed by mass spectrometry to confirm identity

Research examples show consistent detection of SETD7 at 48-50 kDa across multiple studies and antibodies, suggesting this is indeed the correct migration pattern for this protein .

What factors should be considered when selecting and validating SETD7 antibodies for specific applications?

Selecting the appropriate SETD7 antibody requires careful consideration of several factors:

Antibody characteristics to consider:

• Application compatibility: Not all antibodies work equally well across different applications

  • For WB: 24840-1-AP (1:1000-1:5000), OTI2D10 (1:500-2000)

  • For IF/ICC: 24840-1-AP (1:20-1:200), OTI2D10 (1:100)

• Species reactivity: Ensure compatibility with your experimental model

  • Human, mouse, rat: 24840-1-AP

  • Dog, human, monkey, mouse, rat: OTI2D10

• Epitope location: Consider whether the target region is accessible in your application

  • Some antibodies target specific regions (e.g., AA 107-366, AA 257-366)

Validation strategies:

• Positive controls: Use samples known to express SETD7

  • HeLa cells, C6 cells, mouse brain/kidney tissue, NIH/3T3 cells

  • MCF-7 nuclear extract

• Negative controls:

  • siRNA knockdown or CRISPR knockout samples

  • Secondary antibody-only controls for IF/IHC

• Cross-validation:

  • Compare results from multiple antibodies targeting different epitopes

  • Validate using complementary techniques (e.g., mRNA expression, mass spectrometry)

• Citation track record:

  • Some antibodies have established publication records in specific applications:

    • KD/KO (3 publications), WB (8 publications), IHC (2 publications), CoIP (2 publications)

How are SETD7 antibodies being utilized to investigate its role in ROS signaling and oxidative stress?

SETD7 plays an important role in reactive oxygen species (ROS) homeostasis and signaling, with significant implications for inflammatory responses and disease pathogenesis.

Key findings on SETD7 in ROS regulation:

• Inhibition of SETD7 (via siRNA or small molecule inhibitors) counteracts NF-κB-induced oxidative stress and pro-inflammatory cytokine production in macrophages and human bronchial epithelial cells

• SETD7 inhibition elevates mitochondrial antioxidant functions through negative regulation of PPARGC1A and NFE2L2

• SETD7 directly interacts with NFE2L2, affecting the NFE2L2/ARE pathway responsible for antioxidant responses

Methodological approaches using SETD7 antibodies:

• Co-immunoprecipitation: To detect interactions between SETD7 and ROS pathway components like NFE2L2

• Chromatin immunoprecipitation (ChIP): To identify SETD7 binding at promoters of ROS-responsive genes

• Immunofluorescence: To monitor SETD7 translocation in response to oxidative stress

• Western blotting: To assess SETD7 expression changes under oxidative stress conditions

These approaches have revealed that targeting SETD7 might have potential benefits in treating ROS-associated diseases by upregulating multiple antioxidant genes and improving ROS clearance .

What is known about SETD7's role in muscle stem cell expansion, and how can researchers investigate this function?

SETD7 has been identified as a regulator of muscle stem cell (MuSC) expansion with significant implications for cell therapy in muscle diseases:

Key findings:

• Inhibition of SETD7 methyltransferase allows in vitro expansion of myogenic stem cells

• MuSCs expanded with SETD7 small molecule inhibitors show enhanced ability to repopulate the satellite cell niche upon transplantation

• SETD7-inhibited mouse MuSCs demonstrate enhanced therapeutic potential in preclinical models of muscular dystrophy

Contrary to previous assumptions:

• While studies in C2C12 myoblasts suggested SETD7 binds MyoD and regulates myogenic genes through H3K4me1 formation on target promoters, studies in primary MuSCs do not support this mechanism

• Immunostaining and protein fractionation revealed that SETD7 is predominantly cytoplasmic in MuSCs, suggesting a non-chromatin regulatory role

Research approaches using SETD7 antibodies:

• Subcellular localization: Use immunofluorescence and subcellular fractionation with SETD7 antibodies to determine cytoplasmic versus nuclear localization in different muscle cell populations

• Protein-protein interactions: Identify MuSC-specific SETD7 binding partners through co-immunoprecipitation coupled with mass spectrometry

• Expression profiling: Monitor SETD7 expression changes during muscle regeneration and differentiation

• Functional studies: Combine SETD7 inhibition with lineage tracing and antibody-based detection to assess stem cell fate

This research area highlights how SETD7 antibodies have helped challenge established paradigms about this enzyme's function, revealing context-specific roles beyond histone modification.

What reference data should researchers consider when selecting SETD7 antibodies for their studies?

The following comprehensive reference data can guide researchers in selecting appropriate SETD7 antibodies:

SETD7 Protein Characteristics:

• Full Name: SET domain containing (lysine methyltransferase) 7

• Aliases: SET7, SET7/9, SET9, KMT7

• Calculated Molecular Weight: 366 aa, 41 kDa

• Observed Molecular Weight: 48-50 kDa

• GenBank Accession: BC121055

• Gene ID (NCBI): 80854

• UNIPROT ID: Q8WTS6

Common Validated Antibodies and Their Properties:

Storage and Handling Recommendations:

• Store at -20°C

• Stable for one year after shipment

• For 24840-1-AP: Aliquoting is unnecessary for -20°C storage; 20μl sizes contain 0.1% BSA

• For reconstitution of lyophilized antibodies: Add distilled water to a final concentration of about 1 mg/mL

Validated Positive Controls:

• HeLa cells, C6 cells, mouse brain tissue, mouse kidney tissue, NIH/3T3 cells, rat brain tissue (for WB)

• HeLa cells (for IF/ICC)

• MCF-7 nuclear extract

This reference data provides crucial information for experimental design and antibody selection based on specific research needs and applications.