KDM5C Antibody

What are the key applications for KDM5C antibodies in epigenetic research?

KDM5C antibodies are instrumental in multiple research applications crucial for epigenetic studies:

• Western Blot (WB): For detecting KDM5C protein, typically observed at 171-180 kDa molecular weight . Most validated KDM5C antibodies are recommended at dilutions of 1:1000-1:5000 for WB applications .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): For visualizing KDM5C localization primarily in cell nuclei, with recommended dilutions of 1:50-1:500 .

• Chromatin Immunoprecipitation (ChIP): For analyzing KDM5C binding sites throughout the genome, which has revealed that KDM5C demonstrates a strong preference for promoter regions (55% of all KDM5C-enriched sequences) .

• Immunoprecipitation (IP): For studying KDM5C protein interactions and complex formation .

The choice of application should be guided by the specific research question and experimental system being investigated.

How should KDM5C antibodies be stored and handled for optimal performance?

Proper storage and handling are critical for maintaining antibody functionality:

Handling recommendations:

• Avoid repeated freeze-thaw cycles that can degrade antibody quality

• Store in aliquots if frequent use is anticipated

• Most KDM5C antibodies are supplied in buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Allow antibody to equilibrate to room temperature before opening the vial

• Briefly centrifuge before use to collect all solution at the bottom of the tube

For 20μl sizes, note that some products contain 0.1% BSA in their formulation , which may impact certain applications.

What validation criteria should be applied when selecting a KDM5C antibody?

Rigorous validation is essential for reliable research outcomes:

• Reactivity verification: Confirm reactivity with your species of interest. Many KDM5C antibodies show reactivity with human, mouse, and rat samples , but cross-reactivity should be experimentally verified.

• Application-specific validation: Ensure the antibody has been validated for your specific application through:

  • Positive control identification (e.g., HeLa cells, Jurkat cells, and MCF-7 cells typically express detectable levels of KDM5C)

  • Published literature citing the specific antibody for your application

  • Knockout/knockdown validation where available

• Clonality consideration: Choose between:

  • Monoclonal antibodies (e.g., clone 4E7E1) for consistent lot-to-lot reproducibility

  • Polyclonal antibodies for potentially higher sensitivity but with batch variation

• Immunogen mapping: Select antibodies with immunogens matching your region of interest, particularly important when studying KDM5C mutations or truncated variants .

How can KDM5C antibodies be optimized for detecting mutant or truncated KDM5C proteins?

Detection of mutant KDM5C proteins requires careful consideration of antibody epitope location:

Key methodology considerations:

• Epitope mapping relative to mutations: When studying KDM5C mutations, verify the antibody epitope location relative to the mutation site. For instance, in cases like the c.2T>C mutation affecting the translation start codon, antibodies raised against the C-terminus can still detect the N-terminally truncated protein (p.M1_E165del) .

• Multi-antibody approach: Using multiple antibodies targeting different regions of KDM5C provides more comprehensive detection. This is particularly important when analyzing patient samples with potential KDM5C mutations:

  • N-terminal antibodies for C-terminal mutations

  • C-terminal antibodies for N-terminal mutations

  • Domain-specific antibodies when studying particular functional domains

• Western blot optimization: For mutant proteins with altered molecular weights:

  • Wild-type KDM5C appears at ~176 kDa

  • Truncated forms like p.M1_E165del appear at ~156 kDa (20 kDa smaller)

  • Use gradient gels (4-15%) for better resolution

  • Include both wild-type and expected mutant size markers

• Validation in relevant models: Validate antibody performance using:

  • Patient-derived fibroblasts harboring known KDM5C mutations

  • Engineered cell lines with specific KDM5C mutations

  • Knockout cells as negative controls

Research has demonstrated that some KDM5C mutations (e.g., c.3223delG) can lead to complete absence of detectable protein, while others (e.g., c.2T>C) produce truncated proteins that lack functional domains like JmjN and ARID , affecting both detection and function.

What are the critical considerations for using KDM5C antibodies in ChIP-seq experiments?

ChIP-seq with KDM5C antibodies requires specific technical considerations:

• Antibody ChIP-suitability: Not all KDM5C antibodies perform equally in ChIP applications. Select antibodies specifically validated for ChIP-seq , as the fixation process can mask epitopes.

• Fixation optimization:

  • Standard 1% formaldehyde for 10 minutes is often insufficient

  • Test fixation times (5-15 minutes) and formaldehyde concentrations (0.5-2%)

  • Dual crosslinking with DSG (disuccinimidyl glutarate) prior to formaldehyde can improve chromatin binding protein detection

• Sonication parameters:

  • Aim for 200-500 bp fragments

  • Optimize sonication conditions for each cell type

  • Monitor sonication efficiency by agarose gel electrophoresis

• Controls and peak calling strategies:

  • Include input controls and IgG controls

  • For KDM5C particularly, include a spike-in normalization control

  • Use stringent peak calling parameters - research has identified 4,526 high-confidence KDM5C peaks in wild-type cells compared to substantially fewer (576) peaks in mutant cells

• Biological interpretation:

  • KDM5C demonstrates strong preference for promoter regions (55% of all KDM5C-enriched sequences)

  • Integration with H3K4me3 ChIP-seq data is essential for functional interpretation

  • Analysis of KDM5C peaks using pathway databases like KEGG has revealed associations with WNT signaling pathways

How do KDM5C expression levels vary across tissues and cell lines, and how should antibody dilutions be adjusted accordingly?

KDM5C expression varies significantly across tissue and cell types, necessitating experimental adjustments:

Expression patterns:

• Nuclear localization in cytotrophoblasts (CTBs) and extravillous trophoblasts (EVTs)

• Variable expression in cancer cell lines including HeLa, Jurkat, and MCF-7

• Sex-specific differences in expression related to X-chromosome inactivation in females versus single X-chromosome in males

Antibody dilution optimization strategy:

• Pilot titration experiment:

  Tissue/Cell Type	Recommended Starting WB Dilution	Recommended Starting IF Dilution
  High expression (e.g., HeLa)	1:5000	1:500
  Medium expression	1:2000	1:200
  Low expression	1:1000	1:50

• Signal enhancement techniques for low expression systems:

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

  • Signal amplification systems (e.g., TSA)

  • More sensitive detection methods (ECL Plus for WB)

  • Protein concentration methods before analysis

• Quantification and normalization:

  • Always include loading controls appropriate for subcellular fraction

  • Consider spike-in standards for absolute quantification

  • Use recombinant KDM5C standards when absolute quantification is needed

How can researchers distinguish between KDM5C and its paralog KDM5D when studying sex-specific differences?

Distinguishing between KDM5C and KDM5D is particularly important for sex-specific studies:

• Paralog-specific considerations:

  • KDM5C is located on the X chromosome and escapes X-inactivation in females

  • KDM5D is located on the Y chromosome and is male-specific

  • Both proteins have distinct but overlapping functions in histone demethylation

• Antibody selection strategy:

  • Verify antibody specificity against both KDM5C and KDM5D

  • Use epitopes targeting non-conserved regions between paralogs

  • Perform validation in male and female cell lines

  • Include appropriate controls (male cells lacking Y chromosome for KDM5C specificity)

• Experimental design for sex difference studies:

  • The loss of Y chromosome, harboring KDM5D, occurs in most male KDM5C mutant clear cell renal cell carcinomas (ccRCCs)

  • Mutations in KDM5D prevented xenograft tumor formation in male 786-O cells, which was rescued by co-mutation of KDM5C

  • Transcriptional analyses show that KDM5C and KDM5D regulate both overlapping and distinct sets of genes

• Methodological approach:

  • Use combination of antibody-based detection and genetic approaches

  • siRNA/shRNA knockdown validations with paralog-specific reagents

  • CRISPR-Cas9 knockout confirmations when possible

  • Quantitative PCR with paralog-specific primers to confirm target specificity

Research demonstrates that KDM5D in male cells does not function equivalently to the second KDM5C allele in female cells, challenging previous assumptions about these paralogs .

What methodological approaches are most effective for studying KDM5C's role in neurodevelopmental disorders?

KDM5C mutations are implicated in X-linked intellectual disability (ID) and autism spectrum disorder, requiring specialized research approaches:

• Patient-derived cell models:

  • Fibroblasts from patients with KDM5C mutations show reduced KDM5C protein levels

  • iPSC-derived neurons enable studying functional consequences in a relevant cell type

  • Organoid models provide three-dimensional context for neurodevelopmental studies

• Functional assays:

  • Histone demethylase activity assays to assess enzymatic function of mutant KDM5C

  • Transiently transfected mutant KDM5C constructs have shown reduced protein expression, stability, and decreased histone demethylase activities in cells

  • Layer-specific neuronal marker analysis (CTIP2, TBR1) shows reduction in KDM5C knockout models

• Developmental timing considerations:

  • KDM5C's role is critical during specific neurodevelopmental windows

  • Conditional knockout models allow time-specific deletion

  • The canonical WNT signaling pathway has been identified as a potential therapeutic target for intellectual disability, with an unexpected role in cognition during specific developmental windows

• Genotype-phenotype correlations:

  • Patients with KDM5C variations near the C-terminus tend to exhibit autism spectrum disorder in addition to intellectual disability

  • Different mutations produce varying phenotypic severity

  • Novel variations c.2233C>G and c.3392_3393delAG are associated with severe ID, short stature, and facial dysmorphism

• Combined genomic and epigenomic analyses:

  • Integrate RNA-seq data with ChIP-seq and CUT&Tag assays to identify regulatory networks

  • KDM5C binding analysis reveals a strong preference for promoter regions (55% of all KDM5C-enriched sequences)

  • KDM5C regulates genes involved in the WNT signaling pathway

What quality control metrics should be applied when using KDM5C antibodies in quantitative assays?

Rigorous quality control is essential for quantitative applications:

• Assay validation parameters:

  Parameter	Acceptance Criteria	Methodology
  Specificity	Single band at expected MW (171-180 kDa)	Western blot with positive and negative controls
  Sensitivity	Detection limit determination	Serial dilution of recombinant protein or lysates
  Linearity	R² > 0.98 across working range	Standard curve with recombinant protein
  Precision	CV < 15%	Replicate measurements
  Accuracy	85-115% recovery	Spike-in experiments

• Reference standards and normalization:

  • Include recombinant KDM5C standards when possible

  • For relative quantification, select stable reference proteins

  • For ChIP-qPCR, normalize to input and include positive and negative genomic regions

  • Consider spike-in standards for absolute quantification

• Troubleshooting inconsistent results:

  • Verify antibody lot consistency if results change over time

  • Test multiple antibodies targeting different epitopes

  • Consider protein degradation during sample preparation

  • Ensure complete protein extraction from nuclear fractions

• Validation across research platforms:

  • Cross-validate results using orthogonal methods (e.g., MS-based proteomics)

  • Confirm protein-level changes with mRNA expression analysis

  • Use genetic approaches (siRNA, CRISPR) to confirm specificity

How should experiments be designed to study the dual activator/repressor functions of KDM5C?

KDM5C exhibits context-dependent dual functions in gene activation and repression, requiring careful experimental design:

• ChIP-seq and transcriptome integration:

  • Perform parallel H3K4me3 ChIP-seq and RNA-seq in KDM5C wildtype vs. knockout/knockdown systems

  • Categorize genes into KDM5C-activated and KDM5C-repressed groups

  • Identify genomic features associated with each category

  • Research has revealed that KDM5C can regulate both canonical and non-canonical WNT signaling pathways

• Protein complex identification:

  • Co-immunoprecipitation with KDM5C antibodies followed by mass spectrometry

  • Identify context-specific interaction partners in different cell types

  • Compare complex formation in activation versus repression contexts

  • Proximity labeling approaches (BioID/TurboID) to identify transient interactions

• Domain-specific functions:

  • Generate domain deletion constructs (JmjC, ARID, PHD, C5HC2 zinc-finger domains)

  • Rescue experiments with domain mutants in KDM5C-depleted cells

  • Domain-specific ChIP to identify regional binding patterns

  • Catalytic-dead mutants to distinguish enzymatic from scaffolding functions

• Temporal dynamics analysis:

  • Time-course experiments after stimulation/differentiation

  • Compare early versus late KDM5C genomic occupancy

  • Correlate with dynamic changes in histone modifications

  • Inducible systems for temporal control of KDM5C expression

Research in breast cancer has shown that KDM5C can function in both gene transcriptional activation and repression to promote cancer cell growth , highlighting the importance of context in determining KDM5C function.

What are the most effective approaches for studying KDM5C in disease models?

Different disease contexts require tailored experimental approaches:

• Cancer research approaches:

  • KDM5C is commonly mutated in clear cell renal cell carcinomas (ccRCC) in men but rarely in women

  • Mutation of KDM5D in male 786-O cells prevented xenograft tumor formation and was rescued by co-mutation of KDM5C

  • Design experiments accounting for sex differences and Y chromosome loss

  • Test KDM5 inhibitors in preclinical models for therapeutic potential

• Reproductive biology studies:

  • Elevated KDM5C increases recurrent miscarriage risk by preventing trophoblast proliferation and invasion

  • KDM5C is mainly expressed within the nuclei of cytotrophoblasts (CTBs) and extravillous trophoblasts (EVTs)

  • Combine tissue immunohistochemistry with functional trophoblast assays

  • Design in vivo models using systemically delivered KDM5C adenovirus vectors (Ad-KDM5C) which have been shown to promote embryo resorption in mice

• Bone metabolism research:

  • KDM5C controls female bone mass

  • KDM5 inhibitors dose-dependently suppress RANKL-induced osteoclastogenesis

  • Measure metabolic parameters (OCR, ECAR, ATP production) when studying KDM5C in osteoclasts

  • Design experiments testing both mouse models and human primary cells

• Neurodevelopmental disorder investigations:

  • Integrate genomic, transcriptomic, and epigenomic approaches

  • Utilize patient-derived cells, particularly for rare mutations

  • Layer-specific neuronal marker analysis reveals developmental defects in KDM5C models

  • Test potential therapeutic targets like the WNT signaling pathway

How can researchers optimize immunohistochemistry protocols for detecting KDM5C in tissue samples?

Tissue-specific optimization is critical for successful KDM5C immunohistochemistry:

• Sample preparation considerations:

  • Fresh frozen versus FFPE tissue requires different antibody dilutions

  • Antigen retrieval methods: heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) is often optimal

  • Section thickness: 4-5 μm sections typically work best

  • Fixation time affects epitope accessibility and should be standardized

• Protocol optimization strategy:

  • Test multiple antibody concentrations (typically start at 1:50-1:500 range)

  • Compare different blocking reagents (BSA, serum, commercial blockers)

  • Optimize primary antibody incubation (1 hour at room temperature vs. overnight at 4°C)

  • Test signal amplification systems for low-expression tissues

• Tissue-specific considerations:

  • Chorionic villus samples: KDM5C is mainly expressed within the nuclei of CTBs and EVTs

  • Brain tissue: Nuclear staining in neurons with some regional variability

  • Cancer samples: Expression may be heterogeneous within the sample

  • Always include positive control tissues (e.g., HeLa cells embedded in paraffin)

• Validation approaches:

  • Use double immunofluorescence staining with established markers (e.g., CK-7 for trophoblasts)

  • Include tissue from KDM5C knockout models as negative controls

  • Compare multiple antibodies targeting different epitopes

  • Quantify staining intensity using standardized scoring systems or digital image analysis

What strategies are most effective for studying KDM5C and H3K4 methylation dynamics?

Investigating the demethylase activity of KDM5C requires specialized approaches:

• Enzymatic activity assays:

  • In vitro histone demethylase assays with recombinant KDM5C

  • KDM5C specifically demethylates trimethylated and dimethylated but not monomethylated H3K4

  • KDM5C does not demethylate H3K9, H3K27, H3K36, H3K79, or H4K20

  • Use KDM5 inhibitors as controls - they increase H3K4me3 levels in CD14+ monocytes of human peripheral blood mononuclear cells

• ChIP-seq approaches:

  • Parallel KDM5C and H3K4me3 ChIP-seq experiments

  • Analyze genomic regions where KDM5C binding correlates with reduced H3K4me3

  • Time-course experiments to capture demethylation dynamics

  • Combined analysis of RNA-seq, ChIP-seq, and CUT&Tag assays shows that KDM5C overexpression leads to reduction of H3K4me3 on promoters and corresponding downregulation of gene expression

• Advanced epigenomic methods:

  • CUT&Tag for higher resolution profiling of histone modifications

  • Single-cell approaches to capture cellular heterogeneity

  • Mass spectrometry-based histone modification analysis for global quantification

  • Genomic engineering of histone H3 to test modification-specific effects

• Inhibitor-based approaches:

  • KDM5 inhibitors dose-dependently affect biological processes

  • Median inhibitory concentration (IC50) of 5.6 μM has been reported for inhibition of RANKL-induced osteoclastogenesis

  • Monitor dose-dependent increases in H3K4me3 levels as confirmation of target engagement

  • Combine with genetic approaches to confirm specificity

How can inconsistent results with KDM5C antibodies be addressed and resolved?

Troubleshooting strategies for common KDM5C antibody issues:

• Weak or no signal in Western blots:

  • Potential causes and solutions:

    Issue	Solution
    Insufficient protein	Increase loading amount (start with 50-75 μg total protein)
    Inefficient extraction	Use nuclear extraction protocols with detergents
    Protein degradation	Add protease inhibitors freshly; keep samples cold
    Inefficient transfer	Optimize transfer conditions for large proteins (>170 kDa)
    Antibody dilution too high	Try more concentrated primary antibody (1:1000 instead of 1:5000)
    Inadequate detection	Use more sensitive detection systems (ECL Plus)

• Multiple bands or unexpected molecular weight:

  • Potential causes and solutions:

    Issue	Solution
    Protein degradation	Fresh sample preparation; add protease inhibitors
    Alternative splice variants	Verify with RNA analysis; use multiple antibodies targeting different regions
    Post-translational modifications	Use phosphatase treatment to confirm modification status
    Truncated forms	Compare with molecular weight standards; KDM5C mutations can produce truncated proteins (e.g., p.M1_E165del appears at ~156 kDa)
    Non-specific binding	Optimize blocking and washing conditions; use higher antibody dilution

• Inconsistent immunostaining:

  • Standardize fixation methods and times

  • Optimize permeabilization for nuclear antigens

  • Include positive control samples in each experiment

  • Use automated staining systems when available for consistency

  • Standardize image acquisition parameters

• Batch-to-batch variability:

  • Test new antibody lots against reference samples

  • Maintain reference lysates as standards

  • Consider monoclonal antibodies for better reproducibility

  • Document lot numbers and maintain consistent suppliers

What are the best methods for validating KDM5C antibody specificity?

Comprehensive validation approaches ensure reliable results:

• Genetic validation approaches:

  • siRNA/shRNA knockdown followed by Western blot (expected reduction in signal)

  • CRISPR/Cas9 knockout (complete absence of specific signal)

  • Rescue experiments with ectopic expression

  • Quantitative PCR correlation with protein levels

• Cross-reactivity assessment:

  • Test in multiple species if cross-reactivity is claimed

  • Test for cross-reactivity with other KDM5 family members (particularly KDM5D)

  • Peptide competition assays to confirm epitope specificity

  • Pre-adsorption tests with recombinant antigens

• Orthogonal method confirmation:

  • Mass spectrometry validation of immunoprecipitated proteins

  • Correlation with mRNA expression data

  • Independent antibodies targeting different epitopes

  • Multiple application validation (WB, IP, IF, ChIP)

• Context-specific validation:

  • Validation in disease models with known KDM5C alterations

  • Tests in tissues with different KDM5C expression levels

  • Confirmation of subcellular localization (primarily nuclear)

  • Comparison with published literature patterns

KDM5C protein is homologous to three other proteins of the human JARID1 family (JARID1A, JARID1B and JARID1D/KDM5D), sharing several evolutionarily conserved domains that may cause cross-reactivity if not properly validated .

How can KDM5C antibodies be used to study non-histone targets of KDM5C?

Investigating non-canonical functions of KDM5C:

• Co-immunoprecipitation strategies:

  • Use validated KDM5C antibodies for pull-down experiments

  • Analyze by mass spectrometry to identify novel interacting proteins

  • Reciprocal co-IP to confirm interactions

  • Proximity labeling approaches (BioID/TurboID) to identify adjacent proteins

• Functional domain mapping:

  • Generate domain-specific antibodies or tagged constructs

  • Determine which domains mediate non-histone interactions

  • Create domain deletion mutants to test functional consequences

  • Investigate proteins containing lysine methylation sites

• Non-histone methylation targets:

  • Develop methylation-specific antibodies for candidate non-histone targets

  • Use recombinant KDM5C in in vitro demethylation assays

  • Test methylation status of candidates after KDM5C manipulation

  • Apply proteomic approaches to identify global changes in protein methylation

• Subcellular localization studies:

  • Investigate potential cytoplasmic roles using fractionation

  • Co-localization studies with candidate interaction partners

  • Live-cell imaging with fluorescently tagged KDM5C

  • Test localization changes under different cellular conditions

What are the considerations for using KDM5C antibodies in studying the effects of KDM5 inhibitors?

KDM5 inhibitors are emerging therapeutic agents requiring specialized research approaches:

• Target engagement confirmation:

  • Monitor global H3K4me3 levels by Western blot

  • Perform ChIP-seq to identify genomic regions with increased H3K4me3

  • Use H3K4me3 antibodies in parallel with KDM5C antibodies

  • KDM5 inhibitors increase H3K4me3 levels in CD14+ monocytes of human PBMCs

• Functional consequence assessment:

  • KDM5 inhibitors dose-dependently suppress RANKL-induced osteoclastogenesis with an IC50 of 5.6 μM

  • KDM5 inhibition down-regulates key mitochondrial OXPHOS complex proteins and mRNAs

  • Measure cellular processes impacted by inhibition (OCR, ECAR, ATP production)

  • Compare phenotypic effects to genetic KDM5C depletion

• Experimental design considerations:

  • Include dose-response analyses (typically 0.1-10 μM range)

  • Determine time-dependency of effects

  • Address isoform specificity through selective knockdown experiments

  • Test effects across multiple cell types (effects may be context-dependent)

• Combination approaches:

  • Test synergy with other epigenetic modulators

  • Investigate combinations relevant to disease contexts

  • Measure combined effects on transcriptional outputs

  • Develop biomarkers for response prediction