KDM5C Monoclonal Antibody

What is KDM5C and why are monoclonal antibodies against it important in research?

KDM5C is a widely expressed gene that is most highly expressed in the brain and functions as a histone demethylase. It modulates transcriptional activity of genes through demethylation of H3K4, thereby regulating neural development and normal brain function . Mutations in KDM5C are among the most frequent causes of X-linked intellectual disability (XLID). Monoclonal antibodies against KDM5C are crucial research tools because they allow for specific detection of the protein in various experimental contexts, enabling researchers to study its expression, localization, and interactions. These antibodies are particularly valuable when investigating how mutations in KDM5C affect protein function, as demonstrated in recent studies that identified nonsense mutations leading to premature termination codons and subsequent alterations in protein localization and function .

How do I determine the appropriate KDM5C monoclonal antibody for my specific research application?

Selecting the appropriate KDM5C monoclonal antibody depends on several factors related to your experimental goals:

• Epitope recognition: Consider which domain or region of KDM5C your research focuses on. KDM5C contains multiple functional domains including JmjC, zinc finger-C5HC2, JmjN, ARID, and two PHD domains . If studying a specific mutation or domain, select an antibody that recognizes epitopes in that region.

• Experimental application: Different applications require antibodies with specific characteristics:

  • For Western blotting: Choose antibodies validated for denatured protein detection

  • For immunofluorescence: Select antibodies that recognize native protein conformations

  • For immunoprecipitation: Use antibodies with high affinity and specificity

• Species cross-reactivity: Ensure the antibody recognizes KDM5C in your model organism. The study of KDM5C conservation across species indicates high conservation of critical regions like arginine 929 .

• Mutation-specific considerations: If investigating particular mutations like the c.2785 C > T (p.R929X) mutation discussed in recent literature , ensure your antibody's epitope is not affected by the mutation or can specifically distinguish between wild-type and mutant proteins.

What are the recommended protocols for using KDM5C monoclonal antibodies in Western blot experiments?

Based on successful protocols in recent research on KDM5C mutations, the following methodology is recommended for Western blot experiments using KDM5C monoclonal antibodies:

• Sample preparation:

  • Lyse cells on ice using radio immunoprecipitation assay (RIPA) lysis buffer containing proteinase inhibitors

  • Extract total proteins from the supernatant after centrifugation at 12,000 rpm for 15 minutes at 4°C

  • Quantify total protein using a bicinchoninic acid assay

• Protein separation and transfer:

  • Fully denature protein samples with SDS-PAGE protein loading buffer

  • Resolve using 8% SDS-PAGE (appropriate for KDM5C's large molecular weight)

  • Transfer to polyvinylidene fluoride (PVDF) membranes

• Antibody incubation and detection:

  • Block membranes with 5% non-fat milk at 25°C for 1 hour

  • Wash three times with TBST

  • Incubate with primary KDM5C monoclonal antibody at an appropriate dilution (typically 1:1000) at 4°C overnight

  • Wash three times with TBST

  • Incubate with secondary antibody (typically 1:5000 dilution)

  • Visualize using chemiluminescent Western blotting substrate

• Controls and normalization:

  • Include appropriate loading controls (e.g., ACTB/β-actin)

  • For transfection experiments with tagged KDM5C, consider using antibodies against the tag (e.g., anti-EGFP for EGFP-KDM5C fusion proteins) alongside KDM5C-specific antibodies

How can I optimize immunofluorescence protocols using KDM5C monoclonal antibodies?

Optimizing immunofluorescence protocols for KDM5C detection requires attention to several key factors, as demonstrated in recent research studying KDM5C subcellular localization:

• Cell preparation:

  • Plate cells onto coverslips in appropriate culture vessels (e.g., 12-well plates)

  • Seed at an optimal density (approximately 3 × 10⁴ cells per well for HeLa cells)

  • Allow adequate growth time before fixation (36 hours post-transfection when studying expressed constructs)

• Fixation and permeabilization:

  • Carefully aspirate culture medium and wash cells three times with PBS

  • Fix cells with 4% paraformaldehyde in PBS for 20 minutes at room temperature

  • Wash three times with PBS to remove residual paraformaldehyde

  • If needed, permeabilize cells to allow antibody access to intracellular epitopes

• Antibody incubation:

  • Block with appropriate buffer to prevent non-specific binding

  • Incubate with KDM5C monoclonal antibody at optimized dilution

  • Wash thoroughly to remove unbound antibody

  • Incubate with fluorophore-conjugated secondary antibody

• Nuclear counterstaining and mounting:

  • Stain nuclei with DAPI (0.5 μg/ml) for 1 minute

  • Wash three times with PBS to remove excess DAPI

  • Mount slides with appropriate mounting medium

• Imaging considerations:

  • Use confocal fluorescent microscopy at appropriate magnification (40× has been successfully used)

  • When comparing wild-type and mutant KDM5C localization, ensure consistent imaging parameters

How can KDM5C monoclonal antibodies be utilized to investigate nonsense-mediated mRNA decay (NMD) in KDM5C mutation studies?

KDM5C mutations producing premature termination codons (PTCs) can trigger nonsense-mediated mRNA decay (NMD), as observed with mutations like c.2785 C > T (p.R929X) . Investigating this phenomenon requires a sophisticated approach combining antibody techniques with RNA analysis:

• Experimental design strategy:

  • Construct expression plasmids containing wild-type and mutant KDM5C sequences

  • Transfect these constructs into appropriate cell lines (e.g., HeLa or HEK293T)

  • Perform parallel analyses of mRNA levels (by semi-qRT-PCR) and protein levels (by Western blot with KDM5C monoclonal antibodies)

• mRNA analysis workflow:

  • Extract total RNA from transfected cells using Trizol or similar reagents

  • Verify RNA quality (OD260/280 ≈ 2.0)

  • Reverse-transcribe approximately 1 μg of RNA into cDNA

  • Perform semi-quantitative RT-PCR with appropriate primers

• Protein analysis with KDM5C antibodies:

  • Extract proteins from parallel samples

  • Perform Western blotting with KDM5C monoclonal antibodies

  • Compare protein expression levels between wild-type and mutant constructs

  • Normalize to appropriate loading controls

• Integrated data interpretation:

  • Reduced mRNA levels with maintained or increased protein levels (as observed with p.R929X) may indicate complex regulatory mechanisms beyond simple NMD

  • Consider additional factors affecting protein stability, such as the role of ubiquitination pathways (e.g., TRIM11-mediated degradation of KDM5C)

This integrated approach allows researchers to distinguish between mutations that primarily affect mRNA stability through NMD and those that may have additional effects on protein translation, stability, or localization.

What strategies can resolve contradictory results between KDM5C mRNA levels and protein expression detected by monoclonal antibodies?

Researchers may encounter seemingly contradictory results when studying KDM5C mutations, such as decreased mRNA levels alongside increased protein levels. This paradoxical finding was observed with the p.R929X mutation, requiring sophisticated analytical approaches to resolve:

• Mechanistic considerations for discrepant results:

  • Nonsense-mediated mRNA decay (NMD) typically reduces mutant mRNA levels, as observed with several KDM5C mutations

  • Protein stability mechanisms may be independently affected by truncation mutations

  • Domain-specific effects on protein degradation pathways may occur

• Investigation of protein degradation pathways:

  • Examine if the mutation affects known degradation mechanisms for KDM5C

  • Consider testing proteasome inhibitors to determine if protein accumulation is degradation-dependent

  • Investigate specific E3 ubiquitin ligases known to target KDM5C, such as TRIM11, which has been shown to ubiquitinate KDM5C fragment (171-1560aa)

• Domain-specific analysis:

  • Map the truncation relative to known functional and regulatory domains

  • Determine if critical degradation signals or sites are lost in the mutant protein

  • For instance, the p.R929X mutation creates a truncated protein lacking the complete 171-1560aa fragment targeted by TRIM11, potentially explaining its escape from degradation

• Complementary experimental approaches:

  • Pulse-chase experiments to assess protein half-life differences

  • Co-immunoprecipitation studies to examine interactions with degradation machinery

  • Domain-swapping experiments to identify specific regions responsible for altered stability

By systematically investigating these potential mechanisms, researchers can resolve apparent contradictions between mRNA and protein expression data, leading to deeper insights into how KDM5C mutations contribute to disease pathogenesis.

How does epitope selection in KDM5C monoclonal antibodies affect detection of mutant proteins with altered subcellular localization?

The selection of target epitopes in KDM5C monoclonal antibodies is critical when studying mutations that affect protein structure and localization, as seen with the p.R929X mutation that alters nuclear localization:

• Epitope positioning considerations:

  • N-terminal epitopes: Can detect both wild-type and truncated KDM5C, allowing comparison of expression levels

  • C-terminal epitopes: May fail to detect truncated proteins if the epitope is in the deleted region

  • Domain-specific epitopes: Provide information about the presence or absence of specific functional domains

• Subcellular localization analysis strategy:

  • Wild-type KDM5C primarily localizes to the nucleus as expected for a transcription factor

  • Mutant proteins like p.R929X show aberrant cytoplasmic localization

  • Antibodies must maintain specificity in both cellular compartments for accurate comparative studies

• Methodological considerations for detection across compartments:

  • Different fixation protocols may be required for optimal epitope accessibility in nuclear versus cytoplasmic compartments

  • Consider using complementary approaches such as fractionation followed by Western blotting alongside immunofluorescence

  • For fusion proteins, antibodies against protein tags (e.g., EGFP) can provide consistent detection regardless of localization

• Data interpretation framework:

  • Distinguish between genuine changes in localization and artifacts from epitope accessibility

  • Correlate localization changes with structural alterations, such as loss of nuclear localization signals

  • Consider how altered localization affects functional interactions with chromatin and transcriptional machinery

What are the methodological considerations for using KDM5C monoclonal antibodies in chromatin immunoprecipitation (ChIP) experiments?

Chromatin immunoprecipitation (ChIP) with KDM5C monoclonal antibodies enables the investigation of KDM5C's interaction with target genes and chromatin regions, providing crucial insights into its role as a transcriptional regulator:

• Antibody selection criteria for ChIP applications:

  • Epitope accessibility in cross-linked chromatin complexes

  • High specificity to avoid cross-reactivity with related KDM family members

  • Verified ChIP-grade qualification or validation

• Optimization strategies for KDM5C ChIP:

  • Cross-linking conditions: Adjust formaldehyde concentration and fixation time based on KDM5C's chromatin binding characteristics

  • Sonication parameters: Optimize to generate chromatin fragments of appropriate size (typically 200-500 bp)

  • Antibody concentration: Titrate to determine optimal amounts for efficient immunoprecipitation

• Controls and validation approaches:

  • Input chromatin controls to normalize for starting material

  • IgG negative controls to assess non-specific binding

  • Positive controls using primers for known KDM5C target genes

  • Include additional controls when studying mutant KDM5C proteins, such as the p.R929X mutant that fails to localize to the nucleus

• Data analysis considerations:

  • When comparing wild-type and mutant KDM5C binding, account for differences in nuclear localization

  • Consider how altered KDM5C function may affect target gene expression

  • Integrate ChIP data with transcriptome analysis to correlate binding with functional outcomes

This methodological approach is particularly relevant given KDM5C's role in transcriptional regulation through H3K4 demethylation and its impact on neurodevelopmental processes affected in intellectual disability .

How should experiments be designed to compare wild-type and mutant KDM5C detection using monoclonal antibodies?

Designing experiments to effectively compare wild-type and mutant KDM5C requires careful consideration of multiple factors:

• Expression system selection:

  • Plasmid construction: Create comparable expression vectors for wild-type and mutant KDM5C (e.g., pEGFP-KDM5C_WT and pEGFP-KDM5C_MT linked to EGFP C-terminal as used in recent studies)

  • Cell line selection: Choose appropriate cell lines that express necessary cofactors (HeLa and HEK293T cells have been successfully used)

  • Transfection optimization: Use standardized transfection methods (e.g., Lipo8000™ Transfection Reagent) with consistent DNA amounts

• Temporal considerations:

  • Optimal expression time points: Allow sufficient time for expression (36 hours post-transfection has proven effective)

  • Time course studies: Consider examining multiple time points to capture dynamic processes

• Analytical workflows:

  • Parallel analysis pipeline:

    • mRNA analysis via semi-qRT-PCR

    • Protein analysis via Western blotting with KDM5C antibodies

    • Localization studies via immunofluorescence

• Quantification and statistical analysis:

  • Normalization strategies for Western blot data (e.g., using ACTB/β-actin as loading control)

  • Quantitative image analysis for immunofluorescence data

  • Appropriate statistical tests for comparing wild-type and mutant conditions

This comprehensive experimental design enables researchers to systematically evaluate how mutations affect KDM5C at multiple levels, from mRNA stability to protein expression and subcellular localization.

What considerations should be made when designing co-immunoprecipitation experiments with KDM5C monoclonal antibodies?

Co-immunoprecipitation (Co-IP) experiments can reveal protein-protein interactions involving KDM5C, providing insights into its functional complexes and how mutations might disrupt these interactions:

• Buffer optimization for complex preservation:

  • Lysis buffer composition: Use buffers that maintain native protein interactions while efficiently extracting KDM5C

  • Salt concentration: Adjust to balance between extraction efficiency and preservation of protein-protein interactions

  • Detergent selection: Choose mild detergents that solubilize membranes without disrupting protein complexes

• Antibody orientation considerations:

  • Direct approach: Immunoprecipitate with KDM5C monoclonal antibody to pull down interacting partners

  • Reverse approach: Immunoprecipitate with antibodies against suspected interacting partners to detect KDM5C

  • Tagged protein approach: Use anti-tag antibodies (e.g., anti-EGFP for EGFP-KDM5C fusion constructs) for consistent immunoprecipitation

• Controls to establish specificity:

  • Input controls: Analyze a portion of pre-IP lysate to confirm target protein presence

  • IgG controls: Use isotype-matched IgG to identify non-specific binding

  • Competitive peptide controls: Pre-incubate antibody with epitope peptide to block specific binding

• Mutation-specific considerations:

  • For truncated mutants like p.R929X, consider how the mutation affects interaction domains

  • Account for altered subcellular localization (e.g., cytoplasmic versus nuclear) when interpreting interaction data

  • Compare interaction profiles between wild-type and mutant KDM5C to identify disrupted protein complexes

What are the key considerations for quantitative analysis of KDM5C expression using monoclonal antibodies?

Accurate quantification of KDM5C expression levels is essential for understanding how mutations affect protein abundance and function:

For all quantification methods, it's essential to:

• Perform biological and technical replicates

• Apply appropriate statistical analyses

• Consider how mutations (e.g., p.R929X) might affect antibody binding

• Account for differences between endogenous and overexpressed KDM5C levels

How should researchers interpret altered KDM5C subcellular localization detected by monoclonal antibodies?

Changes in KDM5C subcellular localization, as detected by monoclonal antibodies, can provide crucial insights into mutation effects on protein function:

• Interpretation framework for localization changes:

  • Wild-type pattern: KDM5C normally localizes to the nucleus, consistent with its role as a transcriptional regulator and histone demethylase

  • Mutant pattern: Mutations like p.R929X can cause aberrant cytoplasmic localization, indicating loss of nuclear import or retention signals

  • Quantitative assessment: Consider the degree of mislocalization (complete vs. partial) and any cell-type specific effects

• Mechanistic implications of altered localization:

  • Loss of nuclear localization signals: The p.R929X mutation results in a truncated protein lacking nuclear localization residues

  • Domain integrity: Consider how truncation affects the integrity of functional domains

  • Chromatin interaction: Cytoplasmic mislocalization prevents KDM5C from accessing its chromatin targets, functionally equivalent to a complete loss of protein

• Correlation with clinical phenotypes:

  • Severity assessment: Complete mislocalization may correlate with more severe clinical presentations

  • Genotype-phenotype correlations: Compare localization patterns across different mutations to understand phenotypic variability

  • Consider how localization effects contribute to the spectrum of symptoms observed in patients with KDM5C mutations

• Experimental validation approaches:

  • Complementation studies: Test if restoring nuclear localization rescues function

  • Domain mapping: Identify specific regions responsible for proper localization

  • Cell type specificity: Examine if localization defects vary across neural vs. non-neural cell types

What are the common pitfalls in KDM5C monoclonal antibody experiments and how can they be addressed?

Researchers working with KDM5C monoclonal antibodies may encounter several technical challenges that can affect experimental outcomes:

• Cross-reactivity issues:

  • Problem: KDM5C belongs to the KDM5 family with highly homologous members (KDM5A, KDM5B, KDM5D)

  • Solution: Validate antibody specificity using knockout/knockdown controls or by testing against recombinant KDM5 family proteins

  • Implementation: Include KDM5C-deficient samples as negative controls in experiments

• Epitope masking in different applications:

  • Problem: Fixation methods or protein interactions may mask antibody epitopes

  • Solution: Test multiple antibodies targeting different KDM5C epitopes or optimize fixation protocols

  • Implementation: When studying mutants like p.R929X, use antibodies targeting epitopes present in both wild-type and mutant proteins

• Inconsistent detection of mutant proteins:

  • Problem: Truncation mutations may affect antibody binding if epitopes are lost

  • Solution: Use epitope-tagged constructs (e.g., EGFP-KDM5C) for consistent detection

  • Implementation: Compare results from tag-specific and KDM5C-specific antibodies to distinguish expression vs. epitope accessibility issues

• Quantification challenges:

  • Problem: Variations in expression levels between experiments

  • Solution: Include appropriate internal controls and standardize expression systems

  • Implementation: Use semi-qRT-PCR for mRNA and carefully normalized Western blots for protein quantification

How can researchers differentiate between mutation-induced protein degradation and epitope loss when using KDM5C monoclonal antibodies?

Distinguishing between genuine protein degradation and antibody epitope loss is crucial when studying KDM5C mutations:

• Complementary antibody approach:

  • Strategy: Use multiple antibodies targeting different KDM5C epitopes

  • Implementation: Compare detection patterns between N-terminal and C-terminal targeting antibodies

  • Interpretation: Consistent loss of signal across antibodies suggests degradation, while epitope-specific loss indicates epitope disruption

• Tagged protein strategy:

  • Strategy: Generate fusion proteins with epitope tags at termini not affected by the mutation

  • Implementation: Create EGFP-KDM5C constructs as demonstrated in recent studies

  • Interpretation: Detection with anti-tag antibodies provides mutation-independent assessment of protein presence

• mRNA-protein correlation analysis:

  • Strategy: Simultaneously assess mRNA and protein levels

  • Implementation: Perform semi-qRT-PCR and Western blotting from the same samples

  • Interpretation: Reduced protein with normal mRNA suggests degradation; reduced mRNA with reduced protein may indicate nonsense-mediated decay

• Protein stability assays:

  • Strategy: Assess protein half-life using protein synthesis or degradation inhibitors

  • Implementation: Treat cells with cycloheximide (translation inhibitor) or proteasome inhibitors

  • Interpretation: Differential effects of inhibitors between wild-type and mutant KDM5C can reveal degradation mechanisms

This approach was effectively utilized in recent research that revealed the complex relationship between mRNA levels and protein expression for the p.R929X mutation, where reduced mRNA levels were accompanied by increased protein levels, suggesting altered degradation pathways .

What are the best practices for validating novel KDM5C mutations using monoclonal antibodies in patient-derived samples?

Validating newly identified KDM5C mutations in patient samples requires a systematic approach that combines genetic analysis with protein-level investigations:

• Initial genetic and in silico analysis:

  • Confirm mutation through sequencing (e.g., Sanger sequencing following whole exome sequencing)

  • Assess conservation of the affected residue across species

  • Predict functional consequences using computational tools (e.g., Mutation Taster, InterVar)

• Protein expression analysis workflow:

  • Source appropriate patient-derived materials (e.g., lymphoblasts, fibroblasts, or iPSC-derived neural cells)

  • Extract protein using optimized protocols for the specific tissue/cell type

  • Perform Western blotting with KDM5C monoclonal antibodies to assess expression levels

  • Compare with appropriately matched control samples

• Functional validation approaches:

  • Assess subcellular localization using immunofluorescence

  • Evaluate histone demethylase activity through H3K4me3 levels

  • Create recombinant expression constructs mimicking the patient mutation

  • Compare results from patient samples with those from engineered cellular models

• Correlation with clinical phenotype:

  • Document detailed clinical features (e.g., severity of intellectual disability, presence of epilepsy, behavioral phenotypes)

  • Analyze genotype-phenotype correlations across family members

  • Compare with previously reported KDM5C mutations and their associated clinical presentations

The comprehensive approach used to characterize the p.R929X mutation provides an excellent template for validating novel mutations, combining genetic, cellular, and molecular techniques to establish pathogenicity and understand the mechanisms underlying the clinical phenotype .

How can KDM5C monoclonal antibodies be utilized in studying the role of KDM5C in neurodevelopmental disorders beyond intellectual disability?

KDM5C monoclonal antibodies enable investigation of this protein's role in a broader spectrum of neurodevelopmental conditions:

• Developmental expression profiling:

  • Map KDM5C expression patterns across brain regions during development

  • Compare expression in neuronal versus glial populations

  • Assess how KDM5C mutations affect these developmental expression patterns

  • Correlate with the emergence of pathological features observed in conditions like epilepsy, which was present in patients with the p.R929X mutation

• Neural differentiation studies:

  • Examine KDM5C's role in neuronal progenitor differentiation

  • Investigate its contribution to neuronal subtype specification

  • Assess how mutations affect the timely transformation of primary progenitor cells to intermediate progenitor cells, a process regulated by KDM5C through WNT signaling

• Circuit-level investigations:

  • Study KDM5C expression in specific neural circuits implicated in behavioral phenotypes

  • Investigate how mutations affect circuit formation and function

  • Correlate with behavioral manifestations such as hyperactivity and aggressive behavior, which were observed in patients with KDM5C mutations

• Cross-disorder comparative analysis:

  • Compare KDM5C expression and function across multiple neurodevelopmental disorders

  • Investigate shared versus distinct molecular pathways affected by different KDM5C mutations

  • Develop potential biomarkers based on KDM5C status or downstream effects

What are the methodological considerations for using KDM5C monoclonal antibodies in single-cell analysis techniques?

Single-cell techniques offer unprecedented resolution for studying KDM5C expression and function in heterogeneous neural populations:

• Single-cell immunofluorescence optimization:

  • Antibody titration for optimal signal-to-noise ratio

  • Multiplexing strategies for co-detection with cell type markers

  • Image analysis workflows for quantifying expression at single-cell level

• Flow cytometry and cell sorting applications:

  • Cell preparation protocols that preserve KDM5C epitopes

  • Fixation and permeabilization optimization for intracellular staining

  • Gating strategies to identify and isolate KDM5C-expressing cell populations

  • Sorting protocols for downstream molecular analyses

• Single-cell Western blot considerations:

  • Sample preparation for microfluidic single-cell Western platforms

  • Signal amplification strategies for detecting low-abundance KDM5C

  • Quantification approaches for comparing expression across individual cells

• Integration with single-cell sequencing:

  • Protocols for combined protein and RNA analysis from the same cells

  • Computational methods for correlating KDM5C protein levels with transcriptome profiles

  • Strategies for identifying cell populations differentially affected by KDM5C mutations

These advanced single-cell approaches can provide critical insights into the cell type-specific effects of KDM5C mutations, potentially explaining the neurological specificity of symptoms despite KDM5C's widespread expression.

How can KDM5C monoclonal antibodies contribute to developing therapeutic strategies for KDM5C-associated disorders?

KDM5C monoclonal antibodies play crucial roles in developing and evaluating potential therapeutic approaches:

• Target validation applications:

  • Assess engagement of small molecule KDM5C modulators

  • Evaluate effects of potential therapeutics on KDM5C expression, localization, and function

  • Monitor KDM5C-related biomarkers during preclinical and clinical studies

• Rescue strategy assessment:

  • For mutations affecting localization (like p.R929X), evaluate interventions that restore nuclear localization

  • For mutations affecting protein stability, assess therapies that modulate degradation pathways

  • For nonsense mutations triggering NMD, evaluate readthrough compounds that suppress premature termination codons

• Downstream pathway monitoring:

  • Track effects on H3K4 methylation status as a functional readout

  • Monitor expression of KDM5C target genes that regulate neurogenesis, such as WNT pathway components

  • Assess normalization of neuronal differentiation and maturation processes

• Precision medicine applications:

  • Develop assays to classify patients based on molecular mechanisms of their specific KDM5C mutations

  • Match therapeutic approaches to specific mutation types (e.g., mislocalization vs. degradation)

  • Monitor patient-specific responses to therapeutic interventions

By enabling precise monitoring of KDM5C and related pathways, monoclonal antibodies provide essential tools for translating mechanistic understanding into therapeutic development for intellectual disability and related neurodevelopmental disorders.