KMT5C Antibody, HRP conjugated

What is KMT5C and what is its biological function?

KMT5C (also known as SUV420H2) is a histone-lysine methyltransferase that belongs to the Histone-lysine methyltransferase protein family. In humans, the canonical protein consists of 462 amino acid residues with a molecular mass of 52.1 kDa and is primarily localized in the nucleus. Up to 2 different isoforms have been reported for this protein .

KMT5C functions as a histone methyltransferase that specifically methylates monomethylated 'Lys-20' (H4K20me1) and dimethylated 'Lys-20' (H4K20me2) of histone H4 to produce dimethylated 'Lys-20' (H4K20me2) and trimethylated 'Lys-20' (H4K20me3) respectively. Through these modifications, KMT5C plays crucial roles in transcriptional regulation and maintaining genome integrity .

What are the specifications of KMT5C Antibody, HRP conjugated?

KMT5C Antibody, HRP conjugated is a rabbit polyclonal antibody specifically targeting human KMT5C protein. Its specifications include:

This antibody is specifically designed for experimental detection of KMT5C protein in research applications .

How does HRP conjugation benefit KMT5C detection in research applications?

The HRP (Horseradish Peroxidase) conjugation to KMT5C antibody provides significant advantages for detection sensitivity and experimental versatility. The enzymatic activity of HRP catalyzes the oxidation of substrates in the presence of hydrogen peroxide, producing a colorimetric, chemiluminescent, or fluorescent signal depending on the substrate used.

This direct conjugation eliminates the need for secondary antibody incubation, streamlining experimental workflows and reducing potential background signal. The sensitivity of HRP allows for detection of low-abundance KMT5C protein in complex biological samples, which is particularly beneficial when studying histone modifications that may be present at relatively low concentrations .

What are the primary applications of KMT5C Antibody, HRP conjugated?

While various KMT5C antibodies are utilized across multiple applications, the HRP-conjugated variant is particularly suited for specific research methods:

The antibody shows strong reactivity with human samples, making it valuable for translational research between laboratory models and clinical samples .

What is the recommended protocol for Western blot using KMT5C Antibody, HRP conjugated?

For optimal Western blot detection of KMT5C using the HRP-conjugated antibody:

• Prepare protein samples with appropriate lysis buffer containing protease inhibitors

• Separate proteins via SDS-PAGE using 10-12% gels (recommended for the 52.1 kDa KMT5C protein)

• Transfer proteins to a PVDF or nitrocellulose membrane

• Block the membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Dilute the KMT5C Antibody, HRP conjugated at 1:1000 to 1:5000 in blocking buffer

• Incubate the membrane with diluted antibody overnight at 4°C

• Wash the membrane 3-5 times with TBST, 5 minutes each

• Detect using a chemiluminescent substrate compatible with HRP

• Expected band size: approximately 52.1 kDa for the canonical KMT5C protein

For nuclear proteins like KMT5C, it is critical to ensure proper nuclear extraction procedures to maximize yield .

How should the KMT5C Antibody, HRP conjugated be stored and handled to maintain activity?

To maintain optimal activity of the KMT5C Antibody, HRP conjugated:

• Store the antibody at -20°C or -80°C in aliquots to avoid repeated freeze-thaw cycles

• When removing from storage, thaw the antibody on ice

• Prior to use, centrifuge the antibody vial briefly to collect all liquid at the bottom

• Maintain the antibody in the supplied diluent buffer (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) to ensure stability

• Avoid exposure to strong light which may affect HRP activity

• Do not use beyond the expiration date

• Consider adding BSA (0.1-1%) to working dilutions for enhanced stability

Proper storage and handling are crucial as HRP-conjugated antibodies can be susceptible to activity loss if subjected to improper conditions .

How can KMT5C Antibody, HRP conjugated be utilized in epigenetic research?

The KMT5C antibody serves as a valuable tool in epigenetic research due to KMT5C's role in histone H4 lysine 20 methylation:

• Chromatin Dynamics Studies: The antibody can be used to investigate the spatial and temporal distribution of KMT5C at specific genomic loci when combined with ChIP-seq approaches.

• Cell Cycle Regulation Research: Since H4K20 methylation status changes during cell cycle progression, the antibody can track KMT5C recruitment during different cell cycle phases.

• DNA Damage Response Analysis: KMT5C has been implicated in DNA damage repair pathways, and the antibody can be employed to monitor KMT5C recruitment to DNA damage sites.

• Cancer Epigenetics Research: Altered histone methylation patterns are common in cancer, making KMT5C antibody valuable for comparing normal versus malignant tissue samples.

• Interaction Studies: When used in co-IP experiments followed by Western blotting, the HRP-conjugated antibody can directly detect KMT5C in protein complexes .

How does KMT5C activity compare with other histone methyltransferases in experimental systems?

KMT5C functions within a complex network of histone methyltransferases with distinct specificities and roles:

Understanding these differences is crucial when designing experiments targeting specific histone modifications or investigating cross-talk between different histone marks .

What are the considerations for using KMT5C Antibody, HRP conjugated in multiplex immunoassays?

When incorporating KMT5C Antibody, HRP conjugated into multiplex immunoassays, researchers should consider:

• Signal Discrimination: HRP produces a strong signal that may overwhelm weaker signals from other detection systems. Consider using diluted antibody or lower-sensitivity substrates if this occurs.

• Antibody Compatibility: Ensure that other antibodies used in the multiplex assay do not cross-react with KMT5C or interfere with its binding.

• Sequential Detection: For multiple HRP-conjugated antibodies, sequential detection using different substrates with intermediate peroxidase inactivation steps may be necessary.

• Spectral Overlap: When using fluorescent substrates for HRP, consider potential spectral overlap with other fluorophores in the multiplex system.

• Fixation Compatibility: Verify that fixation methods preserve both KMT5C epitopes and other targets in the multiplex system .

What are common challenges in detecting KMT5C and how can they be addressed?

Researchers may encounter several challenges when working with KMT5C antibodies:

Since KMT5C is a nuclear protein with relatively low abundance, nuclear extraction protocols are critical for successful detection .

How can the specificity of KMT5C Antibody, HRP conjugated be validated in experimental systems?

To ensure the specificity of the KMT5C Antibody, HRP conjugated:

• Positive Controls: Include samples with known KMT5C expression (human cell lines like HEK293 or HeLa).

• Negative Controls: Use samples where KMT5C is absent or knockdown/knockout cell lines (through siRNA or CRISPR-Cas9).

• Peptide Competition: Pre-incubate the antibody with excess KMT5C peptide to block specific binding sites.

• Immunoprecipitation-Mass Spectrometry: Confirm that the antibody pulls down KMT5C by mass spectrometry analysis.

• Cross-Reactivity Testing: Test reactivity against closely related proteins like KMT5B (SUV420H1).

• Immunofluorescence Verification: Confirm nuclear localization pattern consistent with KMT5C's known distribution.

• Molecular Weight Verification: Ensure detection at the expected molecular weight (approximately 52.1 kDa) .

What differences should researchers expect when comparing results from KMT5C Antibody, HRP conjugated versus unconjugated variants?

When comparing HRP-conjugated versus unconjugated KMT5C antibodies:

• Sensitivity Differences: HRP-conjugated antibodies often provide enhanced sensitivity due to direct enzyme coupling, but may show reduced epitope binding due to steric hindrance from the HRP molecule.

• Protocol Variations: HRP-conjugated antibodies eliminate the need for secondary antibody incubation, shortening protocols by 1-2 hours.

• Background Considerations: HRP-conjugated antibodies may show different background patterns compared to two-step detection using primary-secondary antibody combinations.

• Storage Stability: HRP-conjugated antibodies typically have shorter shelf lives than unconjugated variants due to potential degradation of the enzymatic component.

• Application Flexibility: Unconjugated antibodies offer greater flexibility across various applications, while HRP-conjugated versions are optimized for specific detection methods like Western blot and ELISA .

How is KMT5C being studied in relation to disease mechanisms?

Recent research has revealed important connections between KMT5C and various disease mechanisms:

• Cancer Biology: Alterations in KMT5C expression and activity have been linked to several cancer types, where dysregulation of H4K20 methylation contributes to genomic instability and aberrant gene expression patterns.

• Neurodevelopmental Disorders: Studies have identified potential roles for KMT5C in neurodevelopment, with implications for certain cognitive disorders associated with epigenetic dysregulation.

• Aging Research: H4K20 trimethylation increases with cellular senescence and aging, making KMT5C a focus in aging research.

• Immune Regulation: Emerging evidence suggests KMT5C may influence immune cell function through epigenetic regulation of key immune response genes.

• Metabolic Disorders: Preliminary research indicates potential connections between histone methylation by enzymes like KMT5C and metabolic pathway regulation .

What are methodological considerations when using KMT5C Antibody, HRP conjugated alongside other histone modification analyses?

When incorporating KMT5C antibody in comprehensive histone modification studies:

• Sequential ChIP Approaches: For studying co-occupancy of KMT5C with other histone marks, sequential ChIP protocols require careful optimization to preserve epitope integrity.

• Normalization Strategies: When quantifying KMT5C levels relative to other histone marks, consistent loading controls and normalization methods are essential.

• Temporal Considerations: Since histone modifications are dynamic, synchronizing cell populations can reduce variability in KMT5C and other histone mark detections.

• Fixation Protocols: Different fixation methods may preferentially preserve certain histone marks while masking others, requiring optimization for multiplex studies.

• Resolution Limitations: When studying KMT5C alongside other histone-modifying enzymes or marks, consider the resolution limitations of antibody-based detection methods versus sequencing-based approaches .

How can researchers effectively combine KMT5C Antibody, HRP conjugated with functional genomics approaches?

To maximize the utility of KMT5C Antibody, HRP conjugated in functional genomics:

• ChIP-seq Integration: Use KMT5C antibody in chromatin immunoprecipitation followed by sequencing to map genome-wide binding sites, complemented by H4K20me2/me3 profiling.

• CRISPR Screening Analysis: Following CRISPR-based KMT5C modulation, the HRP-conjugated antibody provides a sensitive tool for confirming protein-level changes in high-throughput formats.

• Proteomic Interaction Studies: Combine co-immunoprecipitation with mass spectrometry to identify novel KMT5C interaction partners, using the HRP-conjugated antibody for validation studies.

• Single-Cell Applications: For single-cell protein analysis, the sensitivity of HRP detection makes this antibody suitable for detecting KMT5C in limited material.

• Spatial Transcriptomics Correlation: Correlate KMT5C protein localization (detected via the HRP-conjugated antibody) with spatial gene expression data to understand the functional impact of KMT5C-mediated histone methylation .