Mono-methyl-HIST1H3A (R17) Antibody

Basic Research Questions

• What is the Mono-methyl-HIST1H3A (R17) Antibody and what epitope does it specifically recognize?

  The Mono-methyl-HIST1H3A (R17) Antibody is a research tool designed to specifically detect histone H3 when mono-methylated at arginine 17. HIST1H3A encodes Histone H3.1, a core component of nucleosomes that wrap and compact DNA into chromatin, regulating DNA accessibility to cellular machinery . This antibody recognizes the post-translational modification where a single methyl group is attached to the arginine residue at position 17 on histone H3.1, which differs structurally and functionally from unmodified H3, di-methylated H3R17, or other methylation patterns .

• What validated applications are available for Mono-methyl-HIST1H3A (R17) Antibody?

  Based on manufacturer validations, this antibody has been successfully tested in multiple applications with specific recommended dilutions:

  Application	Recommended Dilution Range	Validated Species
  Western Blot (WB)	1:100-1:2000	Human, Mouse, Rat
  Immunohistochemistry (IHC)	1:100-1:250	Human, Mouse, Rat
  Immunocytochemistry/Immunofluorescence (ICC/IF)	1:30-1:500	Human, Mouse, Rat
  ELISA	Varies by manufacturer	Human

  Specific positive controls have been identified, such as mouse colon tissue for IHC applications and HeLa cell lysates for Western blot analysis .

• What is the biological significance of histone H3 R17 mono-methylation?

  Mono-methylation of Histone H3 at arginine 17 is involved in several critical biological processes:

  • Transcriptional regulation by influencing DNA accessibility

  • Chromatin structure modulation

  • DNA repair processes

  • Maintenance of cellular identity through epigenetic programming

  • Signal transduction pathways involving epigenetic mechanisms

  This modification represents one component of the complex "histone code" that dictates genome organization and gene expression patterns across different cell types and developmental stages .

Advanced Research Questions

Methodological Considerations

• What are the optimal protocols for detecting Mono-methyl-HIST1H3A (R17) using immunofluorescence techniques?

  For optimal immunofluorescence detection of Mono-methyl-HIST1H3A (R17), the following validated protocol has proven effective:

  1. Fixation and Permeabilization:

     • Fix cells with 4% paraformaldehyde

     • Permeabilize with 0.1% Triton X-100

  2. Antibody Application:

     • Primary antibody: Apply Mono-methyl-HIST1H3A (R17) antibody at 1:50-1:500 dilution (optimize for specific antibody)

     • For Abcam's EPR17247 clone: Use at 1:2000 dilution

     • Secondary antibody: Use appropriate species-specific fluorophore-conjugated secondary (e.g., Goat anti-rabbit Alexa Fluor 488 at 1:400)

  3. Nuclear Counterstaining:

     • DAPI is recommended for nuclear visualization

     • Include cytoskeletal markers (e.g., tubulin) as needed for cellular context

  4. Controls:

     • Include non-specific IgG controls at equivalent concentrations

     • Consider peptide competition assays using mono-methylated R17 peptides

     • Include positive controls (e.g., cell lines known to express the modification)

  5. Imaging Parameters:

     • Use confocal microscopy for optimal spatial resolution of nuclear signals

     • Maintain consistent exposure settings across samples for quantitative comparisons

• What are the critical factors for successful Western blot detection of Mono-methyl-HIST1H3A (R17)?

  Successful Western blot detection of this histone modification requires careful attention to several technical considerations:

  1. Sample Preparation:

     • Extract histones using specialized acid extraction protocols to enrich for histone proteins

     • Include protease inhibitors and phosphatase inhibitors in lysis buffers

     • For optimal results, use histone preparations rather than whole cell lysates when possible

  2. Gel Electrophoresis:

     • Use 15-18% SDS-PAGE gels for optimal separation of low molecular weight histone proteins

     • Expected molecular weight: approximately 15 kDa

  3. Transfer Conditions:

     • Use PVDF membrane (0.2 μm pore size) for optimal protein retention

     • Short transfer times (60-90 minutes) at lower voltages to prevent small proteins from passing through membrane

  4. Antibody Incubation:

     • Primary antibody dilutions: 1:500-1:2000 for commercial mono-methyl-R17 antibodies

     • From published protocols: 1:1000 dilution for 1 hour at room temperature has shown consistent results

     • Extended blocking times (1-2 hours) with 5% BSA to minimize background

  5. Detection System:

     • Enhanced chemiluminescence (ECL) systems provide sufficient sensitivity

     • Consider fluorescent secondary antibodies for multiplexing with other histone modification antibodies

• How can researchers design peptide competition assays to verify antibody specificity for Mono-methyl-HIST1H3A (R17)?

  Peptide competition assays are essential for confirming antibody specificity, particularly for histone modifications where cross-reactivity with similar epitopes is possible:

  1. Peptide Selection:

     • Synthesize or obtain the following peptides:

       • Target peptide: H3 peptide with mono-methylation at R17

       • Negative control: Unmodified H3 peptide covering the same region

       • Specificity controls: H3 peptides with di-methylation or tri-methylation at R17

       • Cross-reactivity control: H3 peptides with methylation at different residues (e.g., K4, K9, K27)

  2. Competition Protocol:

     • Pre-incubate antibody with a 10-100 fold molar excess of each peptide separately

     • Incubate for 2 hours at room temperature or overnight at 4°C

     • Perform Western blot or immunostaining using the peptide-antibody mixture

  3. Interpretation:

     • Specific binding: Signal should be abolished or significantly reduced only when pre-incubated with the target mono-methyl R17 peptide

     • Complete competition indicates high specificity

     • Partial competition with other methylated R17 peptides may indicate some cross-reactivity

  4. Quantification:

     • Measure signal intensity in control versus competed samples

     • Calculate percent reduction in signal for each peptide competitor

     • Present data in tabular format showing specificity profile across different modifications

Troubleshooting and Optimization

• What are common challenges in detection of Mono-methyl-HIST1H3A (R17) and how can they be addressed?

  Several technical challenges may arise when working with histone modification antibodies:

  Challenge	Potential Cause	Solution
  Weak or no signal	Low abundance of modification	Enrich for histones using acid extraction; Use cell lines with known expression
  High background	Non-specific binding	Increase blocking time/concentration; Optimize antibody dilution; Add 0.1% Tween-20 to wash buffers
  Multiple bands in Western blot	Cross-reactivity with similar modifications	Perform peptide competition assays; Try alternative clone; Use recombinant standards
  Variability between experiments	Protocol inconsistency	Standardize fixation times; Use automated systems; Prepare fresh buffers
  Loss of epitope accessibility	Overfixation	Optimize fixation time; Consider antigen retrieval methods
  Cell type-specific variations	Biological differences in modification levels	Include positive control cell lines; Normalize to total H3 levels

  For immunofluorescence specifically, nuclear staining patterns should be examined carefully, as mono-methyl R17 typically shows nuclear localization with potential enrichment patterns that may correlate with chromatin states .

• How can researchers quantitatively assess Mono-methyl-HIST1H3A (R17) levels across different experimental conditions?

  Quantitative assessment of histone modifications requires rigorous methodological approaches:

  1. Western Blot Quantification:

     • Always run a dilution series of samples to ensure linearity of signal

     • Normalize mono-methyl R17 signal to total histone H3 levels

     • Use digital image analysis software with background subtraction

     • Present data as ratio of modified H3/total H3

  2. Immunofluorescence Quantification:

     • Collect images using identical acquisition parameters

     • Measure nuclear fluorescence intensity using image analysis software

     • Analyze >100 cells per condition for statistical robustness

     • Consider single-cell analysis to capture population heterogeneity

  3. ChIP-seq Approaches:

     • Perform chromatin immunoprecipitation followed by next-generation sequencing

     • Use spike-in controls for normalization between samples

     • Analyze genomic distribution patterns of the modification

     • Compare enrichment at promoters, gene bodies, and regulatory elements

  4. Mass Spectrometry:

     • For absolute quantification, consider using isotopically labeled synthetic peptide standards

     • Compare relative abundance of R17me1 versus other R17 modification states

     • Calculate stoichiometry of the modification across the genome

• How does the choice between monoclonal and polyclonal antibodies affect detection of Mono-methyl-HIST1H3A (R17)?

  The choice between monoclonal and polyclonal antibodies significantly impacts experimental outcomes:

  Characteristic	Monoclonal Antibodies	Polyclonal Antibodies
  Specificity	Highly specific for single epitope Examples: EPR17247 clone , M12477-4	May recognize multiple epitopes around the modification
  Lot-to-lot Consistency	High consistency between batches	Potential variation between lots
  Sensitivity	Sometimes lower sensitivity for rare modifications	Often higher sensitivity due to multiple binding sites
  Background	Generally lower background	May have higher background in some applications
  Applications	Excellent for quantitative applications requiring high specificity	Better for detection of low-abundance modifications
  Cross-reactivity	Less likely to cross-react with similar modifications	Higher potential for cross-reactivity

  For mono-methyl R17 detection, rabbit monoclonal antibodies have demonstrated excellent specificity in multiple applications . When selecting an antibody, researchers should consider:

  • The specific research question (qualitative vs. quantitative)

  • The abundance of the modification in their experimental system

  • The importance of absolute specificity versus sensitivity

  • The particular applications planned (ChIP, IF, WB)

  • The availability of validation data for their specific experimental system