Mono-methyl-HIST1H4A (R67) Antibody

What is Mono-methyl-HIST1H4A (R67) Antibody and what epitope does it recognize?

Mono-methyl-HIST1H4A (R67) Antibody is a polyclonal antibody that specifically recognizes the arginine residue at position 67 when it carries a mono-methylation modification in human Histone H4 protein. The antibody is generated using a peptide sequence surrounding the mono-methylated Arg67 site derived from Human Histone H4 as the immunogen. This antibody is designed for research applications to detect and study this specific post-translational modification of histone proteins .

How does Mono-methyl-HIST1H4A (R67) differ from other histone H4 modification antibodies like Mono-methyl-Histone H4 (K16)?

While both antibodies target post-translational modifications on Histone H4, they recognize different modification sites with distinct biological functions. Mono-methyl-HIST1H4A (R67) specifically detects arginine mono-methylation at position 67, whereas Mono-methyl-Histone H4 (K16) recognizes lysine mono-methylation at position 16. These modifications play different roles in chromatin regulation, with arginine methylation (R67) often associated with transcriptional regulation, while lysine modifications (K16) frequently impact chromatin compaction and DNA accessibility . Additionally, their detection requires specific antibodies with unique epitope recognition profiles - polyclonal for R67 methylation versus monoclonal (clone 3E11) for K16 methylation .

What are the validated applications for Mono-methyl-HIST1H4A (R67) Antibody and their optimal protocols?

The Mono-methyl-HIST1H4A (R67) Polyclonal Antibody has been validated for specific applications including Enzyme-Linked Immunosorbent Assay (ELISA) and Immunocytochemistry (ICC) . For ICC applications, researchers should optimize antibody dilutions based on cell type and fixation methods, typically starting with a dilution range of 1:30-1:200 as recommended for similar histone modification antibodies . When conducting ELISA, proper blocking (typically with 5% BSA or milk proteins) is crucial to minimize background signal. For optimal results, researchers should use positive controls (cells or tissues known to express the modified histone) and negative controls (samples where the modification is absent or blocked). Protocol optimization may include testing different fixation methods, permeabilization conditions, and incubation times to achieve specific signal detection while minimizing background.

How can I validate the specificity of the Mono-methyl-HIST1H4A (R67) Antibody in my experimental system?

Validating antibody specificity for histone modifications requires multiple complementary approaches:

• Peptide competition assays: Pre-incubate the antibody with increasing concentrations of both modified (Mono-methyl-R67) and unmodified peptides before application. A specific antibody will show signal reduction only with the modified peptide.

• Knockout/knockdown controls: Use cells where the enzyme responsible for R67 methylation has been depleted (e.g., PRMT-family enzymes for arginine methylation).

• Specificity factor analysis: Calculate specificity factors (SF) similar to those used for other histone modification antibodies as shown in this table:

Where SF T represents specific binding and SF N represents non-specific binding; a higher SF T/SF N ratio indicates better specificity .

• Cross-reactivity testing: Test against similar modifications (like di-methylation or other arginine methylation sites) to ensure the antibody is truly specific to mono-methylation at R67.

• Multiple detection methods: Confirm results using orthogonal techniques like mass spectrometry to verify the presence and abundance of the modification .

What is the recommended immunoprecipitation protocol for studying chromatin associated with Mono-methyl-HIST1H4A (R67)?

For Chromatin Immunoprecipitation (ChIP) experiments using Mono-methyl-HIST1H4A (R67) Antibody, follow this optimized protocol:

• Crosslinking: Fix cells with 1% formaldehyde for 10 minutes at room temperature to preserve protein-DNA interactions.

• Chromatin preparation: Lyse cells and sonicate chromatin to fragments of 200-500bp, verifying fragment size by gel electrophoresis.

• Pre-clearing: Incubate chromatin with protein A/G beads and non-specific IgG to reduce background.

• Immunoprecipitation: Incubate pre-cleared chromatin with Mono-methyl-HIST1H4A (R67) Antibody (3-5μg) overnight at 4°C. For higher sensitivity, consider implementing the modular antibody approach with enhanced signal detection as described for other histone modifications .

• Washing: Perform stringent washes (low salt, high salt, LiCl, and TE buffer washes) to remove non-specific binding.

• Elution and reversal of crosslinks: Elute immunoprecipitated complexes and reverse crosslinks by heating at 65°C overnight.

• DNA purification and analysis: Purify DNA and analyze by qPCR, sequencing, or other methods.

• Controls: Include input chromatin (non-immunoprecipitated), IgG control (non-specific antibody), and positive control (regions known to be enriched for this modification).

For enhanced sensitivity, consider using signal amplification methods similar to those described for mono-ADP-ribosylation detection, where high-affinity antibodies combined with horseradish peroxidase (HRP) conjugation dramatically increased detection sensitivity .

What are the most common technical challenges when using Mono-methyl-HIST1H4A (R67) Antibody in Western blotting and how can they be addressed?

Common technical challenges with Western blotting for histone modifications like Mono-methyl-HIST1H4A (R67) include:

• Low signal intensity: Enhance detection by:

  • Using optimized protein extraction protocols specifically for histones (acid extraction)

  • Increasing antibody concentration or incubation time

  • Implementing signal enhancement with HRP-conjugated formats that can dramatically improve sensitivity compared to conventional IgG formats

  • Considering immunoprecipitation prior to immunoblotting, which has shown synergistic effects for detection of low-abundance modifications

• High background: Reduce non-specific binding by:

  • Optimizing blocking conditions (5% BSA is often superior to milk for phospho-specific antibodies)

  • Increasing wash duration and stringency

  • Using highly purified primary antibody

  • Ensuring proper antibody dilution (typically 1:1000-1:5000 for Western blot)

• Cross-reactivity: Address by:

  • Performing peptide competition assays with both modified and unmodified peptides

  • Including appropriate controls (positive and negative)

  • Using knockout/knockdown controls for enzymes responsible for the modification

• Inconsistent results: Improve reproducibility by:

  • Standardizing histone extraction procedures

  • Using fresh antibody aliquots

  • Implementing a strictly controlled protocol for all experiments

  • Quantifying signals against total histone H4 levels

How should Mono-methyl-HIST1H4A (R67) Antibody be stored and handled to maintain optimal activity?

For optimal maintenance of Mono-methyl-HIST1H4A (R67) Antibody activity:

• Storage conditions: Store at -20°C or -80°C in small aliquots to prevent repeated freeze-thaw cycles. Most histone modification antibodies are supplied in buffered solutions containing glycerol (typically 50%) and stabilizing agents .

• Working solution preparation: When preparing working dilutions, use fresh, cold buffer (PBS with 0.02% sodium azide, or as recommended by the manufacturer).

• Shelf-life considerations: Even under optimal storage conditions, antibody activity may gradually decrease over time. Validate new lots against previous lots or standards.

• Handling precautions:

  • Avoid repeated freeze-thaw cycles as they can denature antibodies and reduce activity

  • Centrifuge vials briefly before opening to collect solution at the bottom

  • Use sterile techniques when handling to prevent contamination

  • Keep cold when working with the antibody to maintain binding properties

• Buffer compatibility: The antibody is typically supplied in phosphate buffered saline (pH 7.4) with 150mM NaCl, 0.02% sodium azide and 50% glycerol . Ensure that any additives in your experimental buffers don't interfere with antibody binding.

How can Mono-methyl-HIST1H4A (R67) Antibody be used in multiplexed immunofluorescence with other histone modification markers?

Advanced multiplexed immunofluorescence with Mono-methyl-HIST1H4A (R67) Antibody requires careful planning to avoid cross-reactivity while maximizing detection sensitivity:

• Strategic antibody pairing: Combine primary antibodies from different host species (e.g., rabbit anti-Mono-methyl-HIST1H4A with mouse anti-H3K9me3) to enable selective secondary antibody binding.

• Sequential immunostaining: For antibodies from the same host species, implement sequential staining with intermediate blocking steps using fluorophore-conjugated Fab fragments or tyramide signal amplification.

• Spectral unmixing: Utilize spectral imaging and linear unmixing algorithms to separate overlapping fluorescent signals, particularly important when studying co-occurrence of multiple histone modifications.

• Optimization of fixation protocols: Different fixation methods may preferentially preserve certain epitopes; test paraformaldehyde, methanol, and dual fixation approaches to determine optimal conditions for simultaneous detection.

• Signal amplification strategies: Consider implementing modular antibody platforms that enable site-directed labeling with multiple copies of fluorophores or enzymes, which can dramatically increase detection sensitivity as demonstrated for other histone modifications .

• Controls for multiplexed detection: Include single-stained samples for compensation settings and fluorescence minus one (FMO) controls to properly set detection thresholds.

• Quantitative image analysis: Apply specialized image analysis algorithms to quantify co-localization coefficients and relative abundances of different modifications within the same nuclear regions.

What is the role of Mono-methyl-HIST1H4A (R67) in DNA damage response pathways, and how can this antibody be used to study it?

Arginine methylation of histones, including Mono-methyl-HIST1H4A (R67), plays critical roles in the DNA damage response (DDR) pathway. To study this connection:

• Laser microirradiation experiments: Use Mono-methyl-HIST1H4A (R67) Antibody in live-cell imaging following laser-induced DNA damage to track temporal dynamics of this modification at damage sites. This approach could reveal patterns similar to those observed with other modifications like mono-ADPr, which shows a second wave of signaling in the DNA damage response .

• ChIP-sequencing after DNA damage induction: Apply ChIP-seq using the Mono-methyl-HIST1H4A (R67) Antibody before and after treating cells with DNA-damaging agents (e.g., H₂O₂, etoposide, UV) to map genome-wide redistribution of this mark.

• Proximity ligation assays (PLA): Combine Mono-methyl-HIST1H4A (R67) Antibody with antibodies against DNA repair factors to detect their physical proximity at damage sites using PLA technology.

• Protein interaction studies: Use the antibody for co-immunoprecipitation experiments to identify proteins that specifically recognize this modification during DNA damage response.

• Functional studies with PRMT inhibitors: Combine antibody-based detection with pharmacological inhibition of specific protein arginine methyltransferases to determine which enzymes are responsible for damage-induced R67 methylation.

• Correlation with other DNA damage markers: Perform co-staining with γH2AX and other established DDR markers to determine temporal relationships between R67 methylation and known DDR events.

This approach would be similar to studies showing that mono-ADP-ribosylation serves as a recruitment signal for proteins like RNF114 during DNA damage response and telomere maintenance .

How should researchers quantify and normalize Mono-methyl-HIST1H4A (R67) signals across different experimental conditions?

Accurate quantification and normalization of Mono-methyl-HIST1H4A (R67) signals requires systematic approaches:

• Western blot quantification:

  • Use total H4 antibody as loading control and normalize R67 methylation signal to total H4

  • Implement linear range detection by using multiple sample dilutions

  • Apply densitometry with background subtraction for each lane

  • Include a standard curve with known quantities of modified peptide

• Immunofluorescence quantification:

  • Measure nuclear mean fluorescence intensity (MFI) for mono-methyl R67 signal

  • Normalize to total H4 or DAPI signal in the same nucleus

  • Analyze sufficient cell numbers (>100 cells) per condition

  • Use identical acquisition settings across all experimental conditions

• ChIP-qPCR normalization strategies:

  • Calculate percent input for each target region

  • Normalize to a housekeeping gene region that remains stable across conditions

  • Use spike-in chromatin from a different species as an exogenous control

  • Compare to other histone marks as internal controls

• High-throughput data normalization:

  • For ChIP-seq: normalize to input, library size, and account for global changes

  • For proteomics: use label-free quantification with normalization to unmodified peptides

  • For imaging: apply flat-field correction and background subtraction

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Account for biological and technical replicates in analysis

  • Consider using specificity factor analysis similar to that used for other histone antibodies

What are the critical considerations when interpreting changes in Mono-methyl-HIST1H4A (R67) patterns in the context of epigenetic reprogramming studies?

When interpreting changes in Mono-methyl-HIST1H4A (R67) patterns during epigenetic reprogramming:

• Temporal dynamics considerations:

  • Analyze time-course experiments to distinguish between primary and secondary effects

  • Consider the half-life of the modification relative to histone turnover rates

  • Examine correlation with expression changes of arginine methyltransferases and demethylases

• Spatial distribution analysis:

  • Determine genomic localization (promoters, enhancers, gene bodies) of R67 methylation changes

  • Assess co-occurrence with other histone marks to identify combinatorial patterns

  • Map changes relative to chromatin accessibility data (ATAC-seq, DNase-seq)

• Cell heterogeneity impact:

  • Consider single-cell approaches or cell sorting to address population heterogeneity

  • Use immunofluorescence to determine cell-to-cell variability within populations

  • Account for cell cycle effects on histone modifications

• Cross-talk with other modifications:

  • Examine whether changes in R67 methylation affect or are affected by other histone modifications

  • Consider competitive or cooperative relationships between different PTMs at nearby residues

  • Use antibody specificity data to ensure signal represents only the targeted modification

• Functional correlation:

  • Connect R67 methylation changes to functional outcomes (transcription, chromatin compaction)

  • Test causality through targeted modulation of R67 methylation

  • Compare with known epigenetic reprogramming events during development or disease progression

• Technical limitations awareness:

  • Account for antibody specificity limits when interpreting subtle changes

  • Consider epitope masking effects due to neighboring modifications

  • Validate key findings with orthogonal approaches (mass spectrometry)