KDM3A Antibody

What is KDM3A and what is its molecular function?

KDM3A (Lysine Demethylase 3A, also known as JMJD1A or JHDM2A) is a histone demethylase that specifically removes mono- and dimethyl marks from lysine 9 of histone H3 (H3K9me1/2), thereby playing a central role in histone code regulation . It is a member of the JHDM2 histone demethylase family and has a calculated molecular weight of approximately 147.3 kDa, though it is commonly observed at around 150 kDa in Western blot analyses . KDM3A is primarily localized in the nucleus but can also be found in the cytoplasm . Its demethylase activity contributes to transcriptional activation by removing repressive H3K9 methyl marks from gene promoters.

What are the most common applications for KDM3A antibodies?

KDM3A antibodies are primarily used in the following applications:

• Western blotting (WB) - For detecting KDM3A protein expression levels in cell and tissue lysates

• Immunoprecipitation (IP) - For isolating KDM3A protein complexes

• Chromatin Immunoprecipitation (ChIP) - For identifying genomic regions bound by KDM3A

• Immunofluorescence (IF) - For visualizing subcellular localization of KDM3A

• Enzyme-linked immunosorbent assay (ELISA) - For quantitative detection of KDM3A

Most commercially available antibodies recognize human KDM3A, with some also cross-reacting with mouse and rat orthologs .

What controls should be included when validating a new KDM3A antibody?

When validating a new KDM3A antibody, the following controls should be included:

• Positive control: Use cell lines known to express KDM3A (e.g., 293T human embryonic kidney cells, MOLT-4 human acute lymphoblastic leukemia cells)

• Negative control: Include KDM3A knockdown samples using siRNA or shRNA approaches

• Specificity control: Test cross-reactivity with related proteins (KDM3B, KDM3C/JMJD1C) to confirm specificity

• Loading control: Include antibodies against housekeeping proteins (e.g., β-actin) to normalize protein loading

• Subcellular fractionation: Compare detection in nuclear vs. cytoplasmic fractions

• Molecular weight verification: Confirm band appears at expected size (~150 kDa)

• Peptide blocking: If available, perform peptide competition assay to confirm specificity

What are the optimal conditions for Western blot detection of KDM3A?

For optimal Western blot detection of KDM3A:

• Sample preparation:

  • Use RIPA or NP-40 based lysis buffers containing protease inhibitors

  • Load 20-30 μg of whole cell lysate, or 10-20 μg of nuclear extract for enriched detection

• Gel electrophoresis conditions:

  • Use 8% SDS-PAGE gels to properly resolve the 150 kDa KDM3A protein

  • Run under reducing conditions using Immunoblot Buffer Group 1

• Transfer conditions:

  • Transfer to PVDF membrane at low current (250-300 mA) overnight at 4°C for large proteins

• Antibody dilutions:

  • Primary antibody: 1:1000 to 1:2000 dilution (typically 1-2 μg/mL)

  • Secondary antibody: HRP-conjugated anti-species IgG at 1:5000 dilution

• Detection system:

  • Enhanced chemiluminescence (ECL) detection is suitable for most applications

  • Note that degradation or processing products may appear at ~70 kDa

The specific conditions should be optimized for each antibody and sample type, as indicated by manufacturer recommendations.

How should ChIP-Seq experiments with KDM3A antibodies be designed and validated?

Designing and validating ChIP-Seq experiments with KDM3A antibodies requires careful consideration:

• Antibody selection:

  • Choose ChIP-grade antibodies specifically validated for chromatin immunoprecipitation

  • Verify antibody specificity via KDM3A knockdown controls

• Crosslinking and sonication:

  • Use 1% formaldehyde for 10 minutes at room temperature

  • Optimize sonication to obtain chromatin fragments of 200-500 bp

• Experimental controls:

  • Input DNA (pre-immunoprecipitation sample)

  • IgG negative control (non-specific antibody)

  • KDM3A-depleted cells as biological negative control

• Validation approaches:

  • Perform parallel ChIP-qPCR for known KDM3A target regions

  • Include H3K9me1/me2 ChIP to confirm inverse correlation with KDM3A binding

  • Analyze changes in histone marks following KDM3A knockdown

• Data analysis considerations:

  • For KDM3A binding sites, focus on promoter regions of regulated genes

  • Look for correlation with H3K9me1/2 demethylation events

  • Integrate with transcriptome data to identify functional targets

Studies have shown that KDM3A often co-localizes with transcription factors such as estrogen receptor (ER) and androgen receptor (AR) at their target gene promoters .

What troubleshooting approaches can resolve common issues with KDM3A antibody detection?

How can KDM3A antibodies be used to study its role in cancer progression?

KDM3A antibodies can be strategically employed to investigate its role in cancer progression through several methodological approaches:

• Expression analysis in cancer tissues:

  • Immunohistochemistry (IHC) to compare KDM3A levels between normal and malignant tissues

  • Correlation of KDM3A expression with clinical outcomes and patient survival

• Functional studies in cancer models:

  • Combine KDM3A antibodies with knockdown/knockout approaches to validate specificity

  • ChIP-Seq to identify cancer-specific KDM3A targets

  • Analysis of H3K9me1/2 dynamics at oncogene promoters using antibodies against both KDM3A and these histone marks

• Mechanistic studies in specific cancer types:

  • Breast cancer: Investigate KDM3A interaction with estrogen receptor (ER) at target gene promoters using sequential ChIP or co-immunoprecipitation

  • Ewing Sarcoma: Examine KDM3A regulation of metastasis-associated genes like MCAM

  • Prostate cancer: Study co-localization with androgen receptor at target loci

• Therapeutic target validation:

  • Monitor changes in KDM3A levels and activity following treatment with epigenetic inhibitors

  • Combine with cell proliferation, migration, and invasion assays to correlate KDM3A activity with cancer phenotypes

Research has demonstrated that KDM3A regulates genes involved in cell growth, adhesion, and motility across multiple cancer types, making it a potential therapeutic target .

What approaches can be used to study KDM3A post-translational modifications?

Studying KDM3A post-translational modifications requires specialized approaches:

• Phosphorylation analysis:

  • Use phospho-specific antibodies like anti-KDM3A-Y1101p to detect tyrosine phosphorylation

  • Combine with phosphatase treatments as controls

  • Immunoprecipitation with anti-KDM3A followed by immunoblotting with pan-phosphotyrosine antibodies

• Identification of novel modifications:

  • Immunoprecipitate KDM3A followed by mass spectrometry (LC-MS/MS)

  • Compare modification patterns under different cellular conditions

  • Generate and validate site-specific antibodies for newly identified modifications

• Functional implications:

  • Create point mutations at modification sites (e.g., Y1101A)

  • Compare enzymatic activity of wild-type vs. modification-deficient KDM3A

  • Perform ChIP-Seq with wildtype and mutant forms to identify differentially regulated targets

• Signaling pathway analysis:

  • Study kinases responsible for KDM3A modification (e.g., JAK2 for Y1101 phosphorylation)

  • Use kinase inhibitors to modulate KDM3A modification status

  • Examine effects on KDM3A localization, stability, and activity

Research has identified that KDM3A is phosphorylated by JAK2 at tyrosine 1101, with the Y1101 residue being well conserved across species, suggesting functional importance .

How can ChIP-Seq and RNA-Seq be integrated to understand KDM3A-mediated gene regulation?

Integration of ChIP-Seq and RNA-Seq provides powerful insights into KDM3A-mediated gene regulation:

• Experimental design for integration:

  • Perform KDM3A ChIP-Seq, H3K9me1/2 ChIP-Seq, and RNA-Seq in parallel

  • Include KDM3A knockdown/knockout conditions for all assays

  • Consider time-course experiments to capture dynamic regulation

• Data analysis workflow:

  • Identify KDM3A binding sites genome-wide

  • Map H3K9me1/2 changes associated with KDM3A binding

  • Correlate binding events with transcriptional changes from RNA-Seq

  • Perform pathway enrichment analysis of differentially expressed genes

• Functional validation strategies:

  • Select candidate target genes for validation by RT-qPCR

  • Perform ChIP-qPCR at specific loci to confirm KDM3A binding

  • Use luciferase reporter assays with promoter regions of regulated genes

  • Examine co-factor recruitment at KDM3A-regulated promoters

• Advanced insights from integrated analysis:

  • Identify direct vs. indirect KDM3A targets

  • Uncover cell-type specific regulation patterns

  • Map KDM3A cooperation with lineage-specific transcription factors

  • Understand temporal dynamics of gene regulation

Studies combining these approaches have revealed that KDM3A regulates distinct gene clusters in different cancers - estrogen-responsive genes in breast cancer , migration-related genes in Ewing Sarcoma , and androgen-regulated genes in prostate cancer .

How should experiments be designed to study KDM3A in cell differentiation models?

To effectively study KDM3A in cell differentiation models:

• Model system selection:

  • Use established differentiation models like F9 embryonal carcinoma cells

  • Consider embryonic stem cells, where KDM3A maintains pluripotency

  • Compare with lineage-committed cell types

• KDM3A manipulation approaches:

  • Generate stable KDM3A knockdown lines using shRNA

  • Create KDM3A knockout models using CRISPR/Cas9

  • Include wild-type and control vector cells for comparison

• Differentiation experimental design:

  • Induce differentiation using established protocols (e.g., retinoic acid for F9 cells)

  • Monitor morphological changes throughout differentiation

  • Collect samples at multiple time points for comprehensive analysis

• Analysis methods:

  • Assess KDM3A expression changes during differentiation by RT-qPCR and Western blot

  • Examine differentiation markers (e.g., TROMA-1 for primitive endoderm)

  • Analyze expression of key differentiation genes by qRT-PCR

  • Perform ChIP-Seq to identify KDM3A binding sites during differentiation

  • Consider rescue experiments by re-expressing KDM3A or downstream targets

Research has shown that KDM3A is essential for proper differentiation of F9 cells into primitive endoderm, with knockdown cells failing to properly express differentiation markers despite morphological changes .

What strategies can be employed to identify novel KDM3A interaction partners?

To identify novel KDM3A interaction partners:

• Affinity purification approaches:

  • Immunoprecipitation (IP) with anti-KDM3A antibodies followed by mass spectrometry

  • Tag-based approaches (FLAG, HA) with exogenously expressed KDM3A

  • Proximity-based labeling methods (BioID, APEX)

  • Consider both nuclear and cytoplasmic fractions separately

• Validation of interactions:

  • Reciprocal co-immunoprecipitation with antibodies against identified partners

  • GST pull-down assays to test direct interactions

  • Proximity ligation assay (PLA) for visualizing interactions in situ

  • Co-localization studies using immunofluorescence

• Functional characterization:

  • Knockdown of interaction partners to assess effects on KDM3A activity

  • Domain mapping to identify interaction interfaces

  • ChIP-seq for co-localization at genomic loci

  • Transcriptome analysis following partner depletion

• Context-specific interactions:

  • Compare interactomes under different conditions (e.g., hormone stimulation)

  • Analyze cell-type specific interactions

  • Investigate interactions during different cell cycle phases

Studies have identified several important interaction partners for KDM3A including transcription factors like estrogen receptor , androgen receptor , and Ets1 , suggesting context-dependent regulatory mechanisms.

How can researchers distinguish between catalytic and non-catalytic functions of KDM3A?

Distinguishing between catalytic and non-catalytic functions of KDM3A requires sophisticated experimental approaches:

• Generation of catalytically inactive mutants:

  • Create point mutations in the JmjC domain (e.g., H1120G/D1122N)

  • Express wild-type and mutant forms at equal levels

  • Verify loss of demethylase activity using in vitro assays

• Rescue experiments:

  • Deplete endogenous KDM3A using siRNA or shRNA approaches

  • Re-express either wild-type or catalytically inactive KDM3A

  • Compare rescue efficiency for different phenotypes and target genes

• Chromatin analysis:

  • Perform ChIP for H3K9me1/2 marks at KDM3A target genes

  • Compare histone modification patterns with wild-type vs. mutant KDM3A

  • Conduct sequential ChIP to identify factors recruited by KDM3A regardless of its catalytic activity

• Transcriptome analysis:

  • Perform RNA-seq in cells expressing wild-type or catalytically inactive KDM3A

  • Identify genes regulated by KDM3A independent of its demethylase activity

  • Compare with direct binding sites from ChIP-seq data

• Protein domain analysis:

  • Create truncation mutants to isolate functions of different KDM3A domains

  • Examine protein-protein interactions of each domain

  • Assess localization patterns of domain mutants

Research has demonstrated that the catalytic activity of KDM3A is required for estrogen receptor target gene expression and cell growth in breast cancer, indicating that its enzymatic function is essential for at least some of its biological roles .

How should researchers interpret apparent discrepancies in KDM3A molecular weight?

When interpreting discrepancies in KDM3A molecular weight:

• Expected vs. observed molecular weight:

  • KDM3A has a calculated molecular weight of approximately 147.3 kDa

  • In Western blots, it is commonly observed at approximately 150 kDa

  • A band at approximately 70 kDa may represent degradation or processing products

• Potential explanations for discrepancies:

  • Post-translational modifications (phosphorylation , potential ubiquitination, etc.)

  • Alternative splicing resulting in different isoforms (at least two isoforms are known)

  • Protein processing or degradation during sample preparation

  • Differences in gel systems and molecular weight standards

• Validation approaches:

  • Use multiple KDM3A antibodies targeting different epitopes

  • Include KDM3A knockdown/knockout controls to confirm specificity

  • Perform immunoprecipitation followed by mass spectrometry for definitive identification

  • Compare detection patterns across different cell lines and tissues

• Experimental considerations:

  • Use gradient gels for better resolution of high molecular weight proteins

  • Include appropriate molecular weight markers covering the expected range

  • Consider using phosphatase treatment to eliminate phosphorylation-based shifts

The observation of both full-length (~150 kDa) and partial (~70 kDa) KDM3A bands is documented in the literature and should be interpreted in the context of the specific experimental conditions .

What criteria should be used to validate KDM3A as a therapeutic target in cancer research?

Validating KDM3A as a therapeutic target in cancer research requires multiple lines of evidence:

• Expression analysis in clinical samples:

  • Evaluate KDM3A expression in tumor vs. normal tissues across large patient cohorts

  • Correlate expression with clinical outcomes and survival

  • Stratify analysis by cancer subtypes and stages

• Functional dependency studies:

  • Perform KDM3A knockdown/knockout in multiple cancer cell lines

  • Evaluate effects on proliferation, apoptosis, migration, and invasion

  • Test in both conventional and therapy-resistant models

  • Validate in 3D organoid cultures and xenograft models

• Mechanistic understanding:

  • Identify KDM3A-regulated oncogenic pathways through integrated genomic analyses

  • Determine if KDM3A regulates established cancer hallmarks

  • Evaluate effects on therapy response genes

  • Assess synthetic lethality interactions

• Therapeutic potential assessment:

  • Develop and test small molecule inhibitors of KDM3A catalytic activity

  • Compare genetic vs. pharmacological inhibition phenotypes

  • Evaluate potential synergies with established therapies

  • Assess possible toxicities through studies in normal cells

Research has shown that KDM3A regulates estrogen receptor signaling in breast cancer , promotes metastasis in Ewing Sarcoma through MCAM regulation , and modulates androgen receptor activity in prostate cancer , supporting its potential as a therapeutic target across multiple cancer types.

How can researchers resolve contradictory findings about KDM3A function across different studies?

When encountering contradictory findings about KDM3A function:

• Systematic comparison of experimental conditions:

  • Cell type and tissue context differences (e.g., embryonic vs. differentiated, normal vs. cancer)

  • Knockdown/knockout methodology (transient vs. stable, partial vs. complete)

  • Antibody specificity and epitope locations

  • Experimental timing and dynamic processes

• Molecular context considerations:

  • Expression levels of KDM3A cofactors and interaction partners

  • Presence of compensatory mechanisms (e.g., other KDM family members)

  • Cell-type specific transcription factor landscapes

  • Epigenetic background and chromatin accessibility

• Technical validation approaches:

  • Reproduce key experiments using multiple methodologies

  • Use complementary gain- and loss-of-function approaches

  • Validate antibody specificity through knockout controls

  • Test critical findings across multiple cell lines/models

• Integrative interpretation strategies:

  • Develop context-specific models of KDM3A function

  • Consider distinct roles in different biological processes

  • Integrate data across multiple omics platforms

  • Acknowledge limitations and biological complexity