SETD7 Antibody

What is SETD7 and why is it important in cellular research?

SETD7 (SET domain containing lysine methyltransferase 7) was the first lysine methyltransferase discovered to specifically monomethylate lysine-4 of histone H3. It plays central roles in transcriptional activation of genes such as collagenase and insulin . Beyond histone modification, SETD7 has a broad target specificity, methylating non-histone proteins including transcriptional regulators like p53, TAF10, ER, p65, STAT3, and others . Its function in both epigenetic regulation and protein modification makes it crucial for studying gene expression, inflammation, oxidative stress response, and various disease mechanisms.

How do I choose between polyclonal and monoclonal SETD7 antibodies?

The choice depends on your experimental goals:

Polyclonal SETD7 antibodies:

• Recognize multiple epitopes on the SETD7 protein

• Often provide higher sensitivity for applications like Western blot

• Available from multiple vendors (Thermo Fisher, Proteintech, St John's Laboratory)

• Better for detecting native proteins or when protein conformation may be altered

Monoclonal SETD7 antibodies:

• Recognize a single epitope with high specificity

• Provide consistent lot-to-lot reproducibility

• Examples include OriGene's OTI2D10 clone

• Preferable for applications requiring minimal background or when distinguishing between closely related proteins

For initial characterization studies, a polyclonal antibody might provide better detection, while follow-up studies requiring higher reproducibility might benefit from monoclonal antibodies.

What species reactivity should I consider when selecting a SETD7 antibody?

When selecting a SETD7 antibody, verify the species reactivity based on your experimental model:

Always validate the antibody for your specific application and species, as reactivity may vary based on the immunogen sequence and antibody production method .

What are the optimal applications for SETD7 antibodies?

SETD7 antibodies can be used in multiple applications, with varying levels of optimization:

Note that the expected molecular weight of SETD7 is approximately 40-41 kDa, but it typically appears at 48-50 kDa in Western blots due to post-translational modifications .

How can I optimize Western blotting conditions for SETD7 detection?

For optimal Western blot detection of SETD7:

• Sample preparation: Use RIPA or NP-40 buffer with protease inhibitors

• Protein loading: Load 20-40 μg of total protein per lane

• Gel percentage: 10-12% SDS-PAGE is optimal for the 41-50 kDa range

• Antibody dilution: Start with 1:1000 for most antibodies, then optimize

• Expected band size: Look for bands at 48-50 kDa (observed) versus the calculated 40.5-41 kDa size

• Positive controls: HeLa cells, NIH/3T3 cells, mouse brain tissue, and C6 cells all express detectable levels of SETD7

• Blocking: 5% non-fat milk in TBST is generally effective, though BSA may be preferred for phospho-specific detection

Always include appropriate loading controls and validate specificity using SETD7 knockdown or knockout samples when possible.

How can I effectively use SETD7 antibodies in immunofluorescence studies?

For successful immunofluorescence detection of SETD7:

• Fixation method: 4% paraformaldehyde (PFA) for 15-20 minutes at room temperature

• Permeabilization: 0.1-0.3% Triton X-100 for 5-10 minutes

• Blocking: 5% normal serum (matching secondary antibody host) with 1% BSA

• Primary antibody dilution: Start with 1:100 for most SETD7 antibodies

• Incubation: Overnight at 4°C or 1-2 hours at room temperature

• Secondary antibody: Use fluorophore-conjugated antibodies at 1:200-1:500

• Nuclear counterstain: DAPI or Hoechst

• Expected localization: SETD7 is primarily nuclear but can also be detected in cytoplasm

HeLa cells have been successfully used as a positive control for SETD7 immunofluorescence staining .

How should SETD7 antibodies be stored and handled to maintain activity?

Proper storage and handling are crucial for antibody longevity and performance:

• Storage temperature: Store at -20°C for long-term preservation

• Aliquoting: For antibodies in glycerol solutions, aliquoting may be unnecessary. For lyophilized antibodies, reconstitute and aliquot to avoid freeze-thaw cycles

• Working dilutions: Store at 4°C for up to 2 weeks; for longer periods, add sodium azide (0.05-0.1%)

• Reconstitution: For lyophilized antibodies, reconstitute with distilled water to approximately 1 mg/mL

• Stability: Most antibodies remain stable for 12 months from date of receipt when properly stored

• Buffer composition: Most SETD7 antibodies are provided in PBS with glycerol (50%) and possibly BSA (0.5%) and sodium azide (0.02%)

Avoid repeated freeze-thaw cycles as they can degrade antibody quality and performance.

What positive and negative controls should be used when working with SETD7 antibodies?

Proper controls are essential for validating antibody specificity:

Positive controls for SETD7 detection:

• Cell lines: HeLa, NIH/3T3, C6 cells

• Tissues: Mouse brain, mouse kidney, rat brain

• Overexpression systems: HEK293T cells transfected with SETD7 expression vector

Negative controls:

• Primary antibody omission

• Isotype control (IgG from same species as the primary antibody)

• SETD7 knockdown (siRNA) or knockout (CRISPR/Cas9) samples

• Blocking peptide competition (pre-incubate antibody with immunogen peptide)

Incorporating both positive and negative controls ensures confidence in the specificity of your SETD7 staining pattern.

How can I validate SETD7 antibody specificity for my experimental system?

Validating antibody specificity is crucial for reliable results:

• Western blot analysis:

  • Confirm band at expected molecular weight (48-50 kDa observed)

  • Compare multiple antibodies targeting different epitopes

  • Test in multiple cell lines/tissues known to express SETD7

• Genetic approaches:

  • siRNA knockdown should reduce signal proportionally to knockdown efficiency

  • CRISPR/Cas9 knockout should eliminate specific signal

  • Overexpression should increase signal intensity

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide

  • Signal should be significantly reduced or eliminated

• Cross-reactivity assessment:

  • Test in species or samples where the antibody is not expected to react

  • Check for non-specific bands or staining patterns

Why might I observe multiple bands in Western blots when using SETD7 antibodies?

Multiple bands may appear for several reasons:

• Post-translational modifications: SETD7 can be subject to phosphorylation and other modifications that alter migration

• Isoforms: Alternative splicing may generate different SETD7 variants

• Degradation products: Proteolytic cleavage during sample preparation

• Cross-reactivity: Some antibodies may recognize related SET domain-containing proteins

• Non-specific binding: Especially with polyclonal antibodies

To address multiple bands:

• Use freshly prepared samples with protease inhibitors

• Try different antibodies targeting different epitopes

• Include positive controls with known SETD7 expression

• Perform validation using SETD7 knockdown/knockout

• Note that the expected size (40-41 kDa) often appears as 48-50 kDa in gels

How can I improve SETD7 signal detection in immunohistochemistry applications?

For enhanced IHC signal:

• Antigen retrieval optimization:

  • Try heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Optimize retrieval duration (10-20 minutes)

• Antibody concentration:

  • Test serial dilutions (1:150-1:500 is recommended for most SETD7 antibodies)

  • Extend primary antibody incubation to overnight at 4°C

• Detection system:

  • Try polymer-based detection systems for enhanced sensitivity

  • Consider tyramide signal amplification for low-abundance targets

• Fixation considerations:

  • Limit fixation time to preserve epitope accessibility

  • Try frozen sections if formalin-fixed samples yield poor results

• Background reduction:

  • Increase blocking time/concentration

  • Add 0.1-0.3% Triton X-100 to reduce non-specific binding

  • Include avidin/biotin blocking if using biotin-based detection

What might cause inconsistent results between different lots of SETD7 antibodies?

Lot-to-lot variability can arise from:

• Production variables:

  • Changes in immunization protocols

  • Natural variation in animal immune responses

  • Different purification batches

• Storage and handling:

  • Degradation during shipping or storage

  • Freeze-thaw cycles affecting antibody stability

To address variability:

• Purchase larger quantities of a single lot for long-term studies

• Validate each new lot against previous lots

• Consider monoclonal antibodies for greater consistency

• Maintain standardized protocols for antibody handling

• Include positive controls in each experiment for normalization

How can SETD7 antibodies be used to study protein-protein interactions and methylation targets?

SETD7 interacts with numerous proteins as both binding partners and methylation substrates:

• Co-immunoprecipitation (Co-IP):

  • Use SETD7 antibodies to pull down SETD7 and identify interacting proteins

  • Several SETD7 antibodies have been validated for Co-IP applications

  • Reverse Co-IP with antibodies against suspected partners can confirm interactions

• Proximity ligation assay (PLA):

  • Detect SETD7 interactions with specific proteins in situ

  • Requires antibodies from different host species for SETD7 and partner protein

• Methylation target identification:

  • Use anti-methyl lysine antibodies after SETD7 overexpression

  • Combine with mass spectrometry to identify methylated residues

  • Verify with in vitro methylation assays using recombinant SETD7

• Known interactors and targets:

  • Transcription factors: p53, TAF10, ER, P65, STAT3, Foxo3

  • Other proteins: cGAS, IPF1/PDX-1, Rb, Mypt, Tat

  • Recognition motif: [KR]-[STA]-K sequence in substrate proteins

Studies have demonstrated direct interaction between SETD7 and NFE2L2, suggesting SETD7's role in regulating antioxidant responses .

How can SETD7 antibodies be utilized to investigate its role in oxidative stress and inflammation?

SETD7 plays significant roles in ROS signaling and inflammatory responses:

• Oxidative stress studies:

  • Use SETD7 antibodies to monitor protein levels and localization during oxidative stress

  • Combine with inhibitors or knockdown to assess functional outcomes

  • Research shows SETD7 inhibition can counter NF-κB-induced oxidative stress and elevate mitochondrial antioxidant functions

• Inflammation research applications:

  • Monitor SETD7-mediated methylation of NF-κB p65 during inflammatory activation

  • Track SETD7 recruitment to inflammatory gene promoters using ChIP assays

  • Assess effects of SETD7 inhibition on pro-inflammatory cytokine production

• SETD7-NFE2L2 pathway:

  • Investigate interaction between SETD7 and NFE2L2 using Co-IP

  • Study effects on antioxidant gene expression

  • SETD7 inhibition upregulates multiple antioxidant genes via the NFE2L2/ARE pathway

• Potential therapeutic targeting:

  • Use antibodies to validate SETD7 inhibitor efficacy in cellular models

  • Monitor methylation status of SETD7 targets during inhibitor treatment

  • Assess downstream effects on ROS clearance and inflammatory signaling

What methodological approaches can be used to study SETD7's epigenetic functions using available antibodies?

To investigate SETD7's role in epigenetic regulation:

• Chromatin Immunoprecipitation (ChIP):

  • Use SETD7 antibodies to identify genomic binding sites

  • Combine with H3K4me1-specific antibodies to correlate SETD7 binding with histone methylation

  • Follow with sequencing (ChIP-seq) for genome-wide analysis

• Sequential ChIP (Re-ChIP):

  • First IP with SETD7 antibody, then with antibodies against transcription factors

  • Identifies genomic regions where SETD7 co-localizes with specific factors

• Gene expression correlation:

  • Combine SETD7 knockdown/overexpression with RT-qPCR or RNA-seq

  • Use SETD7 antibodies to verify protein levels

  • Correlate with H3K4me1 levels at specific genomic regions

• Methyltransferase activity assays:

  • Immunoprecipitate SETD7 using specific antibodies

  • Perform in vitro methyltransferase assays with recombinant histones

  • Detect H3K4me1 levels using specific antibodies

SETD7 plays a central role in transcriptional activation of genes like collagenase and insulin, and is recruited by transcription factors such as IPF1/PDX-1 to specific promoters .

How are SETD7 antibodies being used to understand its role in disease models?

SETD7 antibodies are enabling research into multiple disease contexts:

• Cancer research:

  • Monitoring SETD7-mediated methylation of p53 (Lys-372), which stabilizes p53 and increases transcriptional activation

  • Studying SETD7's interactions with estrogen receptor (ER) in hormone-responsive cancers

  • Investigating potential roles in cell cycle regulation through Rb methylation

• Metabolic disorders:

  • Examining SETD7's role in insulin gene transcription via IPF1/PDX-1 recruitment

  • Assessing SETD7 regulation of metabolic gene expression

• Inflammatory diseases:

  • Investigating SETD7's contribution to NF-κB signaling and pro-inflammatory cytokine production

  • Studying potential as therapeutic target in inflammatory conditions

• Oxidative stress-related conditions:

  • Exploring SETD7 inhibition as a means to improve ROS clearance in stress conditions

  • Examining interactions with NFE2L2 and effects on antioxidant gene expression

SETD7 antibodies provide essential tools for tracking protein levels, localization, and target methylation in these disease models.

What methodological challenges exist in studying SETD7 methyltransferase activity using antibodies?

Studying SETD7's methyltransferase activity presents several technical challenges:

• Detecting low-abundance methylation:

  • Methylation is often substoichiometric

  • May require enrichment strategies or highly sensitive detection methods

  • Pan-methyl-lysine antibodies often have limited sensitivity

• Target-specific methylation detection:

  • Generation of methylation-site-specific antibodies is technically challenging

  • Validation requires extensive controls including methylation-deficient mutants

  • Cross-reactivity with related methylation sites may occur

• Temporal dynamics:

  • Methylation events may be transient or context-dependent

  • Requires careful experimental timing and appropriate stimulation conditions

• Competition with other modifications:

  • Methylation sites may overlap with other PTMs (phosphorylation, acetylation)

  • May require sequential IP approaches to resolve modification patterns

To address these challenges, researchers often combine antibody-based detection with mass spectrometry validation, in vitro enzymatic assays, and genetic manipulation of SETD7 or its targets.