KMT5C Antibody, Biotin conjugated

What is KMT5C and why is it important in epigenetic research?

KMT5C (also known as SUV420H2) is a histone-lysine methyltransferase that specifically methylates monomethylated 'Lys-20' (H4K20me1) and dimethylated 'Lys-20' (H4K20me2) of histone H4 to produce dimethylated and trimethylated 'Lys-20' (H4K20me3) respectively. This enzyme plays a critical role in transcriptional regulation and genome integrity maintenance. Its importance in epigenetic research stems from its involvement in chromatin organization and gene expression control, making it a valuable target for studies on cellular differentiation, cancer biology, and response to therapy .

What are the key advantages of using biotin-conjugated KMT5C antibodies over unconjugated versions?

Biotin-conjugated KMT5C antibodies offer several methodological advantages:

• Enhanced sensitivity through signal amplification via the strong biotin-streptavidin interaction

• Greater flexibility in experimental design with compatibility across multiple detection systems

• Reduced background in multi-color immunofluorescence experiments

• Improved stability during long-term storage compared to directly labeled fluorescent antibodies

• Versatility in applications including ELISA, immunofluorescence, and chromatin immunoprecipitation followed by sequencing (ChIP-seq)

What are the optimal fixation and permeabilization procedures when using biotin-conjugated KMT5C antibodies for immunofluorescence?

For optimal results with biotin-conjugated KMT5C antibodies in immunofluorescence:

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.3% Triton X-100 in PBS for 10 minutes

• Block with 5% normal serum (from the same species as the secondary antibody) with 0.1% Triton X-100 for 1 hour

• Incubate with biotin-conjugated KMT5C antibody at 1:50 to 1:100 dilution overnight at 4°C

• Detect using streptavidin-conjugated fluorophores (FITC, Texas Red, etc.)

• Use DAPI for nuclear counterstaining

This protocol has been validated in multiple cell types including embryonic cells as demonstrated in published immunofluorescence analyses .

How can I quantitatively measure H4K20me3 levels using biotin-conjugated KMT5C antibodies in cells with different genetic backgrounds?

Quantifying H4K20me3 levels requires a rigorous approach:

• Western Blot Quantification:

  • Use biotin-conjugated KMT5C antibody followed by streptavidin-HRP

  • Include β-actin or total H4 as loading controls

  • Perform densitometry analysis using ImageJ or similar software

  • Normalize H4K20me3 signal to loading control

• Immunofluorescence Quantification:

  • Implement parallel staining of WT and mutant samples

  • Use identical acquisition parameters (exposure time, gain)

  • Quantify nuclear signal intensity using CellProfiler or similar software

  • Normalize to nuclear area or DAPI intensity

  • Analyze ≥100 cells per condition

• In-cell Western Analysis:

  • Plate equal cell numbers in 96-well format

  • Perform fixation and antibody incubation in-plate

  • Use GAPDH as endogenous control

  • Scan with appropriate imaging system

  • Calculate relative H4K20me3 levels

This multi-method approach enables robust quantification across different genetic backgrounds, as demonstrated in studies comparing H4K20me3 levels in KMT5C wild type and mutant cell lines .

What are the critical steps for successful ChIP-qPCR experiments using biotin-conjugated KMT5C antibodies to identify H4K20me3-enriched genomic regions?

For successful ChIP-qPCR with biotin-conjugated KMT5C antibodies:

• Chromatin Preparation:

  • Crosslink cells with 1% formaldehyde for exactly 10 minutes

  • Quench with 125mM glycine

  • Sonicate to achieve fragments of 200-500bp (verify by gel electrophoresis)

• Immunoprecipitation:

  • Pre-clear chromatin with protein G beads

  • Incubate chromatin with biotin-conjugated KMT5C antibody overnight at 4°C

  • Capture antibody-chromatin complexes with streptavidin-coated magnetic beads

  • Include IgG control for background determination

• qPCR Design:

  • Design primers targeting regions of interest, such as promoters or enhancers

  • Include primers for known H4K20me3-enriched regions as positive controls

  • Include primers for regions devoid of H4K20me3 as negative controls

  • Calculate fold enrichment as the ratio of immunoprecipitated DNA to input DNA, relative to IgG control

• Data Analysis:

  • Present data as fold enrichment of regions pulled down by H4K20me3 antibody relative to IgG

  • Compare enrichment between experimental conditions (e.g., WT vs. mutant)

  • Apply appropriate statistical tests (one-way ANOVA with Dunnett's multiple comparison test)

This approach has been successfully applied to identify H4K20me3-enriched regions in regulatory elements, such as those upstream of MET gene .

How does loss of KMT5C function affect EGFR inhibitor sensitivity, and how can biotin-conjugated KMT5C antibodies be used to study this mechanism?

Loss of KMT5C function has been demonstrated to confer resistance to EGFR tyrosine kinase inhibitors (TKIs) including erlotinib, gefitinib, afatinib, and osimertinib in non-small cell lung cancer (NSCLC) cell lines. Biotin-conjugated KMT5C antibodies can be instrumental in investigating this mechanism through:

• Monitoring H4K20me3 Levels:

  • Compare H4K20me3 levels in sensitive versus resistant cell lines using Western blot and immunofluorescence

  • Correlate H4K20me3 levels with GI50 values for various EGFR inhibitors

  • Track changes in H4K20me3 during acquired resistance development

• ChIP-seq Analysis:

  • Identify genome-wide changes in H4K20me3 distribution following KMT5C loss

  • Uncover potential regulatory elements affected by KMT5C deficiency

  • Correlate with expression changes in resistance-associated genes

• Target Gene Identification:

  • Studies have shown that loss of KMT5C leads to upregulation of MET and MKK3, contributing to EGFR-TKI resistance

  • H4K20me3 marks on regulatory elements like LINC01510 can be detected using biotin-conjugated KMT5C antibodies

  • The methylation status of these regions correlates with gene expression changes

This approach provides insights into the epigenetic mechanisms of drug resistance, as exemplified by studies showing that KMT5C transcript levels are downregulated in tumors post-treatment with osimertinib in NSCLC patients .

What experimental design would effectively demonstrate the relationship between KMT5C activity, H4K20 trimethylation, and cancer drug resistance?

An effective experimental design would include:

• Baseline Analysis:

  • Measure KMT5C expression and H4K20me3 levels across a panel of cancer cell lines with varying drug sensitivities

  • Use biotin-conjugated KMT5C antibody for immunofluorescence and Western blot analysis

  • Correlate findings with drug sensitivity data (GI50 values)

• Genetic Manipulation:

  • Generate KMT5C knockout or knockdown models using CRISPR-Cas9 or siRNA

  • Create KMT5C overexpression models in sensitive cell lines

  • Validate alterations in H4K20me3 levels using biotin-conjugated antibodies

  • Assess changes in drug sensitivity using dose-response assays

• Pharmacological Inhibition:

  • Treat cells with KMT5B/C inhibitors (e.g., A-196)

  • Confirm reduction in H4K20me3 levels

  • Evaluate changes in drug sensitivity

  • Compare effects with genetic manipulation

• Mechanistic Investigation:

  • Perform RNA-seq to identify differentially expressed genes

  • Use ChIP-seq with biotin-conjugated KMT5C antibodies to map H4K20me3 distribution

  • Identify candidate genes regulated by KMT5C

  • Validate candidates through targeted knockdown/overexpression

• Clinical Correlation:

  • Analyze KMT5C expression and H4K20me3 levels in patient samples pre- and post-treatment

  • Correlate findings with treatment response and survival outcomes

This comprehensive approach has been successfully employed to demonstrate that KMT5C loss induces MET and MKK3 overexpression in EGFR-mutant cell lines, contributing to therapy resistance .

What are common sources of background signal when using biotin-conjugated KMT5C antibodies, and how can they be mitigated?

These approaches have been validated across multiple cellular systems and applications including immunofluorescence in embryonic cells and Chlamydomonas cells .

How can researchers optimize ChIP protocols when using biotin-conjugated KMT5C antibodies to detect low-abundance H4K20me3 marks?

Optimization strategies for detecting low-abundance H4K20me3 marks include:

• Increase Cell Input:

  • Start with at least 10⁷ cells for each immunoprecipitation

  • Scale buffer volumes accordingly while maintaining antibody concentration

• Crosslinking Optimization:

  • Dual crosslinking approach: 2 mM disuccinimidyl glutarate (DSG) for 45 minutes followed by 1% formaldehyde for 10 minutes

  • This preserves protein-protein interactions more effectively than formaldehyde alone

• Sonication Refinement:

  • Optimize sonication conditions to achieve consistent 200-300bp fragments

  • Verify fragment size by agarose gel electrophoresis

  • Consider enzymatic fragmentation (MNase) as an alternative

• Immunoprecipitation Enhancement:

  • Extend incubation time with antibody to 16-20 hours at 4°C with gentle rotation

  • Use a sequential ChIP approach for highly specific enrichment

  • Add carrier proteins (e.g., sheared salmon sperm DNA) to reduce non-specific binding

• Signal Amplification:

  • Implement biotin-streptavidin signal amplification systems

  • Consider using specialized ChIP-grade streptavidin beads

  • Reduce bead volume to concentrate the signal

• qPCR Sensitivity:

  • Design highly efficient primers (90-110% efficiency)

  • Increase PCR cycles (up to 45) for low-abundance targets

  • Use nested PCR approach for extremely low signals

This optimized approach has been successfully applied to detect H4K20me3 marks on chromatin regions with varying enrichment levels, as demonstrated in studies examining the regulation of genes like MET by KMT5C .

How do the functions of KMT5C differ from other histone methyltransferases, and what experimental approaches with biotin-conjugated antibodies can distinguish these differences?

KMT5C has distinct functions compared to other histone methyltransferases:

Experimental approaches to distinguish these differences using biotin-conjugated antibodies:

• Sequential ChIP (Re-ChIP):

  • First IP with biotin-conjugated KMT5C antibody

  • Elute complexes and perform second IP with antibodies against other HMTs

  • Analyze co-occupancy of different HMTs at specific genomic loci

• Co-Immunoprecipitation:

  • Use biotin-conjugated KMT5C antibodies for pull-down

  • Probe for interaction partners

  • Compare interactome with other HMTs

• Histone Modification Profiling:

  • Perform Western blots with antibodies against various histone marks (H4K20me1/2/3, H3K9me3, H3K27me3)

  • Compare modification patterns in WT, KMT5C-mutant, and other HMT-mutant cells

  • Assess cross-talk between different histone modifications

• Inhibitor Studies:

  • Compare effects of KMT5B/C-specific inhibitors (e.g., A-196) with inhibitors of other HMTs

  • Measure changes in global histone modification levels

  • Assess differential effects on cellular phenotypes and gene expression

These approaches have been instrumental in distinguishing the specific roles of KMT5C in processes like EGFR inhibitor resistance, which appears to be uniquely regulated by this particular HMT through specific target genes .

What are the most effective strategies for multiplexing biotin-conjugated KMT5C antibodies with other epigenetic markers in mass cytometry or multi-parameter imaging?

For effective multiplexing of biotin-conjugated KMT5C antibodies with other epigenetic markers:

• Panel Design for Mass Cytometry (CyTOF):

  • Use metal-conjugated streptavidin (e.g., Sm-149-streptavidin) to detect biotin-conjugated KMT5C antibodies

  • Select metals with minimal signal overlap for other epigenetic markers

  • Include both activating (H3K4me3, H3K27ac) and repressive (H3K9me3, H3K27me3) marks

  • Add cell cycle markers (e.g., Ki-67) for cell state context

  • Example panel design:

  Target	Metal Tag	Antibody Type	Function
  KMT5C	Sm-149 (via streptavidin)	Biotin-conjugated primary	H4K20 methyltransferase
  H4K20me3	Nd-142	Direct metal-conjugated	KMT5C product
  H3K9me3	Eu-151	Direct metal-conjugated	Heterochromatin mark
  H3K27me3	Gd-160	Direct metal-conjugated	Polycomb-mediated silencing
  H3K4me3	Yb-172	Direct metal-conjugated	Active promoters
  Ki-67	Ir-191	Direct metal-conjugated	Proliferation marker

• Multiplex Immunofluorescence:

  • Use far-red fluorophore-conjugated streptavidin to detect biotin-conjugated KMT5C antibody

  • Apply tyramide signal amplification (TSA) for low-abundance targets

  • Implement sequential antibody labeling and stripping protocol:
    a. Apply first primary antibody
    b. Detect with appropriate secondary
    c. Fix signal with 4% PFA
    d. Strip remaining antibodies with glycine-SDS buffer (pH 2.5)
    e. Repeat for each target

  • Use spectral unmixing algorithms to separate overlapping fluorophores

• Imaging Mass Cytometry:

  • Prepare FFPE or frozen tissue sections

  • Apply biotin-conjugated KMT5C antibody followed by metal-tagged streptavidin

  • Include antibodies against cell type-specific markers

  • Analyze spatial relationships between KMT5C, its histone mark, and other epigenetic features

  • Implement neighborhood analysis to identify cellular microenvironments

• CODEX (CO-Detection by indEXing) Approach:

  • Conjugate biotin-KMT5C antibody with unique DNA barcodes

  • Apply all barcoded antibodies simultaneously

  • Detect sequentially through repeated cycles of complementary fluorescent oligo hybridization

  • This enables visualization of 40+ targets on the same sample

These multiplexing strategies have been applied successfully in epigenetic research to understand the complex interplay between different histone modifications and their regulatory enzymes .

How can biotin-conjugated KMT5C antibodies be used to investigate the role of H4K20 methylation in metabolism and adipose tissue regulation?

Biotin-conjugated KMT5C antibodies offer valuable tools for investigating H4K20 methylation in metabolic regulation:

• Adipocyte Differentiation Studies:

  • Track changes in H4K20me3 levels during adipocyte differentiation

  • Perform ChIP-seq with biotin-conjugated KMT5C antibodies at different time points

  • Correlate H4K20me3 distribution with expression of adipogenic genes

  • Compare patterns in brown versus white adipose tissue

• Metabolic Phenotyping:

  • Research has shown that Suv420h histone methyltransferases (including KMT5C/SUV420H2) regulate PPAR-γ and energy metabolism

  • Use biotin-conjugated KMT5C antibodies to investigate H4K20me3 marks on metabolic gene promoters

  • Correlate with physiological parameters in metabolic disease models

• Tissue-Specific Analysis:

  • Perform immunohistochemistry on adipose tissue sections using biotin-conjugated KMT5C antibodies

  • Quantify nuclear H4K20me3 levels in different cell populations

  • Compare normal versus metabolically challenged tissues (high-fat diet, diabetes models)

• Response to Environmental Stimuli:

  • Studies have demonstrated that Suv420h enzymes respond to environmental stimuli

  • Investigate H4K20me3 pattern changes following cold exposure, caloric restriction, or exercise

  • Correlate with mitochondrial function and brown adipose tissue activation

• Intervention Studies:

  • Use A-196 inhibitor to modulate KMT5B/C activity in metabolic tissues

  • Monitor subsequent changes in H4K20me3 patterns and gene expression

  • Assess metabolic parameters (oxygen consumption, energy expenditure)

This research approach can reveal how KMT5C-mediated H4K20 methylation contributes to metabolic regulation, as suggested by studies showing that Suv420h histone methyltransferases regulate PPAR-γ and energy metabolism .

What are the critical considerations when using biotin-conjugated KMT5C antibodies for studying H4K20me3 dynamics during cell cycle progression?

When studying H4K20me3 dynamics during cell cycle progression with biotin-conjugated KMT5C antibodies, researchers should consider:

• Cell Synchronization:

  • Implement precise synchronization methods (double thymidine block, nocodazole arrest, etc.)

  • Verify synchronization efficiency by flow cytometry

  • Collect cells at defined time points throughout the cell cycle

• Fixation Timing and Method:

  • Rapid fixation is critical to capture transient states

  • Use formaldehyde fixation (1-4%) for 10 minutes at room temperature

  • Avoid methanol fixation which can extract histones

• Dual Labeling Strategy:

  • Combine biotin-conjugated KMT5C antibody with cell cycle markers:

    • G1: Cyclin D1 or CDT1

    • S: EdU incorporation or PCNA

    • G2: Cyclin B1

    • M: Phospho-histone H3 (Ser10)

  • Use different fluorophores to distinguish cell cycle stages

• Quantitative Analysis:

  • Implement high-content imaging to analyze thousands of cells

  • Classify cells by cycle phase based on marker expression

  • Quantify nuclear H4K20me3 intensity for each phase

  • Calculate relative changes in H4K20me3 throughout the cycle

• Dynamic ChIP Studies:

  • Perform ChIP-seq with biotin-conjugated KMT5C antibodies at different cell cycle phases

  • Compare H4K20me3 distribution patterns across the genome

  • Identify cell cycle-regulated H4K20me3 domains

• Live-Cell Imaging Considerations:

  • For real-time dynamics, use cell lines expressing fluorescently tagged histones

  • Complement with fixed-cell time course using biotin-conjugated KMT5C antibodies

  • Correct for potential cell cycle variability through single-cell analysis

• Technical Controls:

  • Include IgG controls for each cell cycle phase

  • Use KMT5C knockout cells as negative controls

  • Include total H4 staining to normalize for histone content changes

This methodological approach enables precise characterization of H4K20me3 dynamics throughout the cell cycle, providing insights into the temporal regulation of chromatin structure and its impact on genome stability and gene expression .