Histone H3R26me1 Antibody

What is Histone H3R26me1 and what biological significance does it have?

Histone H3R26me1 is a post-translational modification where arginine 26 on histone H3 is monomethylated. This modification is part of the complex "histone code" that regulates chromatin structure and gene expression. Histone H3 is one of the core histone proteins (H2A, H2B, H3, and H4) that form the nucleosome, the basic unit of chromatin packaging in eukaryotes.

Arginine methylation can occur in three forms: monomethylation (me1), asymmetric dimethylation (me2a), and symmetric dimethylation (me2s). Each form potentially plays distinct roles in gene regulation. While H3R26me1 is less extensively characterized than some other histone marks like H3K4me3 or H3K27me3, arginine methylation generally is associated with both transcriptional activation and repression, depending on the specific residue modified and the cellular context.

The arginine methylation of histones is catalyzed by protein arginine methyltransferases (PRMTs), and these modifications can influence recruitment of specific effector proteins that affect chromatin compaction, nucleosome positioning, and ultimately gene expression patterns.

What commercially available H3R26me1 antibodies exist and what are their specifications?

Several commercial suppliers offer H3R26me1 antibodies with the following specifications:

All these antibodies are:

• Generated using synthetic monomethylated peptides corresponding to the region surrounding R26 of human histone H3

• Purified by affinity chromatography

• Formulated in PBS with 0.02% sodium azide, 50% glycerol, pH 7.3

• Unconjugated, requiring appropriate secondary antibodies for detection

The recommended working dilutions typically range from:

• Western blot: 1:500-1:2000

• Immunofluorescence/Immunocytochemistry: 1:50-1:200

• ELISA: 1 μg/ml

What applications can H3R26me1 antibodies be used for?

Based on manufacturer validations and typical histone antibody applications, H3R26me1 antibodies can be used for:

• Western Blotting (WB): For detecting and quantifying H3R26me1 levels in cell or tissue lysates. The expected molecular weight is approximately 17 kDa on SDS-PAGE gels (versus the calculated MW of 15-16 kDa) .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): For visualizing the nuclear localization and distribution patterns of H3R26me1 in fixed cells.

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of H3R26me1 in solution.

• Immunohistochemistry (IHC): For examining H3R26me1 distribution in tissue sections, particularly with paraffin-embedded samples (IHC-P).

While not specifically validated for all commercially available H3R26me1 antibodies, other potential applications include:

• Chromatin Immunoprecipitation (ChIP): Many histone PTM antibodies are used to identify genomic regions enriched for specific modifications. Some H3 antibodies are specifically validated as "ChIP Grade" .

• ChIP-sequencing (ChIP-seq): For genome-wide mapping of H3R26me1 distribution.

• Flow Cytometry: For quantifying H3R26me1 levels in individual cells.

• CUT&RUN or CUT&Tag: For higher resolution mapping of H3R26me1 genomic localization.

How should H3R26me1 antibodies be stored and handled to maintain optimal performance?

Based on manufacturer recommendations, proper storage and handling includes:

• Storage temperature:

  • Long-term: Store at -20°C

  • Short-term (up to 2 weeks): May be stored at 2-8°C

• Aliquoting: Divide into small aliquots upon receipt to avoid repeated freeze-thaw cycles, which can degrade antibody quality and reduce performance .

• Formulation: Typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability .

• Thawing protocol: Thaw on ice or at 4°C, avoid rapid temperature changes.

• Handling precautions:

  • Use sterile technique to prevent microbial contamination

  • Avoid vortexing, which can cause protein denaturation

  • Centrifuge briefly before opening the vial to collect liquid at the bottom

• Shelf-life: Typically 12 months from date of receipt when stored properly, though this may vary by manufacturer .

• Transportation: Should be shipped with cold packs or on dry ice to maintain low temperature.

What specificity concerns exist for H3R26me1 antibodies?

Like many histone PTM antibodies, H3R26me1 antibodies may exhibit several specificity concerns that researchers should be aware of:

• Cross-reactivity with similar modifications: Antibodies may recognize the same modification (monomethylation) at different arginine residues in histones, particularly if they share similar surrounding sequences.

• Inability to distinguish methylation states: Some antibodies cannot effectively discriminate between unmethylated, monomethylated, and dimethylated forms of the same residue. For example, search result states: "many antibodies to histone PTMs fail to specifically recognize their intended targets."

• Off-target recognition: Research has shown that "A significant number of histone PTM antibodies that were screened demonstrated cross-reactivity with unintended modifications" . A specific example from search result showed a H3K9me3 antibody cross-reacting with "H3K27me3, H3K23me3, and H3K18me3."

• Influence of neighboring modifications: The presence of other post-translational modifications near R26 can significantly affect antibody binding (discussed in detail in section 2.2).

• Lot-to-lot variation: Particularly for polyclonal antibodies, significant variation can exist between production lots. Search result notes that "currently available anti-histone antibodies are mostly polyclonal, and hence each lot of antibody is a different product that needs to be extensively validated prior to use."

To address these specificity concerns, researchers should:

• Validate each antibody lot using appropriate controls

• Use peptide competition assays to confirm specificity

• Consider validating findings with multiple antibodies from different sources or with orthogonal techniques like mass spectrometry

How do neighboring modifications influence H3R26me1 antibody recognition?

The "neighboring modification effect" is a critical consideration for histone PTM antibodies. Research has demonstrated that modifications adjacent to the target epitope can dramatically affect antibody binding:

"We observed that a monoclonal antibody widely used against H3K4me3 is perturbed mainly by modification at Histone H3 arginine 2 (H3R2)... Contrastingly, a widely used polyclonal antibody from Millipore was negatively influenced by H3T6 phosphorylation" .

For H3R26me1 antibodies specifically, modifications that might affect binding could include:

• Lysine 23 acetylation (H3K23ac) or methylation (H3K23me)

• Lysine 27 methylation (H3K27me) or acetylation (H3K27ac)

• Serine 28 phosphorylation (H3S28ph)

The practical implications of this phenomenon are significant. For example, research has shown that "A polyclonal antibody raised against H3S10 phosphorylation showed a statistically significant reduction in binding to peptides also modified at H3K9" . This means that changes in one modification (H3K9 acetylation) could be misinterpreted as changes in another modification (H3S10 phosphorylation) due to antibody behavior.

To address this issue:

• Use peptide arrays containing combinatorial modifications to test antibody specificity

• Consider the biological context and likely co-occurring modifications when interpreting results

• When possible, use complementary approaches (e.g., mass spectrometry) to confirm the presence of H3R26me1

What are the advantages and limitations of polyclonal versus monoclonal H3R26me1 antibodies?

Polyclonal Antibodies:

• Advantages:

  • Recognize multiple epitopes on the antigen, potentially increasing sensitivity

  • Less affected by small changes in the epitope or sample preparation

  • Generally provide stronger signals in applications like Western blot

• Limitations:

  • Significant lot-to-lot variation requiring validation of each lot

  • Higher background and potential cross-reactivity

  • Limited reproducibility for quantitative comparisons across long-term studies

Monoclonal Antibodies:

• Advantages:

  • Consistent between lots with minimal variation

  • Typically higher specificity for a single epitope

  • Better suited for quantitative comparisons across experiments

• Limitations:

  • May have lower sensitivity than polyclonals

  • More affected by small changes in epitope structure due to fixation or denaturation

  • A single epitope may be masked in certain applications or contexts

Search result highlights an emerging alternative: "we produced recombinant antibodies directed to the trimethylated lysine residues of histone H3 with high specificity and affinity and no lot-to-lot variation." These recombinant antibodies aim to combine the advantages of monoclonals (consistency) with potentially higher affinity.

When choosing between antibody types for H3R26me1 detection, researchers should consider:

• The importance of lot-to-lot consistency for long-term projects

• The specific application (ChIP may benefit from polyclonal antibodies' ability to recognize multiple epitopes)

• The validation data available for each specific antibody clone or lot

How can researchers validate the specificity of H3R26me1 antibodies?

Comprehensive validation of histone PTM antibodies is essential. Based on search results , and , the following validation methods are recommended:

• Peptide Arrays: Test antibody binding against a panel of modified and unmodified histone peptides.

  • Example implementation: "We recently established a quantitative peptide immunoprecipitation assay that determines the dissociation constant" .

• Peptide Competition Assays: Pre-incubate the antibody with excess H3R26me1 peptide before application.

  • "Additionally, peptide competition assays verified the interaction between the H3K14 antibodies and the H3K36ac peptide" .

• Knockout/Knockdown Controls: Use cells lacking the enzyme responsible for establishing H3R26me1.

  • "To assess the specificity of these antibodies for H3K27 methylation, parallel ChIP-Seq experiments were performed in ES cells lacking H3K27 methylation due to genetic deletion of the EED core subunit of the PRC2 histone methyltransferase complex" .

• Western Blot Analysis: Confirm the antibody detects a band of the expected size (approximately 17 kDa).

  • "Western blot validation: Confirm the antibody detects a band of the expected size (approximately 17 kDa) and that this band is absent or reduced in appropriate negative controls" .

• ChIP-Seq Validation: Compare results from multiple antibodies targeting the same modification.

  • "Analysis of wild-type mouse embryonic stem cells showed a strong correlation of read distribution between the two antibodies" .

• Semi-synthetic Nucleosome IPs: Use DNA-barcoded mononucleosomes with defined modifications.

  • "We tested two H3K27me3 antibodies and two H3K79me2 antibodies using both native and cross-linking IP conditions" .

• Orthogonal Techniques: Validate findings using mass spectrometry or other antibody-independent methods.

Importantly, validation should be application-specific:

• For Western blot: Include histone extraction controls and loading controls

• For ChIP: Include input controls and IgG controls

• For IF/IHC: Include peptide competition controls and secondary-only controls

What strategies can mitigate lot-to-lot variation issues with H3R26me1 antibodies?

Lot-to-lot variation is a significant challenge with histone PTM antibodies. Based on search results and , several strategies can help mitigate this issue:

• Purchase larger quantities of validated lots: When a thoroughly validated lot is identified, purchase enough for the entire project to ensure consistency.

• Perform comprehensive lot-specific validation: Each new lot should be validated using the same controls and standards as previous lots to ensure comparable performance. This includes peptide arrays, Western blot analysis, and application-specific tests.

• Use recombinant antibodies when available: As mentioned in search result , recombinant antibodies offer "high specificity and affinity and no lot-to-lot variation."

• Create internal reference standards: Maintain aliquots of positive control samples (cell lysates or peptides) that can be used to calibrate new antibody lots.

• Use multiple antibodies from different sources: When possible, confirm key findings with independent antibodies targeting the same modification.

• Document lot numbers in publications: Record and report the specific lot numbers used in experiments to allow proper reproduction of results.

• Use online resources: Consult databases like "The Histone Antibody Specificity Database" mentioned in search result to check validation data for specific antibody lots.

• Consider synthetic alternatives: Some researchers are developing synthetic binding reagents (aptamers, nanobodies) that may offer more consistent alternatives to traditional antibodies.

What is the optimal protocol for Western blotting with H3R26me1 antibodies?

Based on the manufacturers' recommendations and general principles for histone Western blotting, the following protocol is suggested:

Sample Preparation:

• Extract histones using an acid extraction method (e.g., 0.2N HCl) to enrich for basic proteins

• Quantify protein concentration using Bradford or BCA assay

• Load 10-20 μg of histone extract or 30-50 μg of whole cell lysate

Gel Electrophoresis:

• Use 15-18% SDS-PAGE gels or 4-12% Bis-Tris gradient gels

• Run at 200V for 35-40 minutes under reducing conditions

Transfer:

• Transfer to PVDF or nitrocellulose membrane (0.2 μm pore size recommended)

• Transfer at lower voltage (30V) for longer time (60-70 minutes) for efficient transfer of small proteins

Immunoblotting:

• Block with 5% BSA in TBST (not milk, which contains proteins that can interfere with histone antibody binding)

• Incubate with primary H3R26me1 antibody at recommended dilution (1:500-1:2000)

• Incubate overnight at 4°C

• Wash extensively with TBST (at least 3 x 10 minutes)

• Incubate with HRP-conjugated secondary antibody (typically anti-rabbit at 1:5000-1:20000)

• Develop using ECL substrate and image

Controls to Include:

• Total H3 antibody on a parallel blot or after stripping and reprobing

• Peptide competition control

• Cell line with known H3R26me1 status (positive control)

• Loading control (typically a housekeeping protein for whole cell lysates)

Expected Results:
The H3R26me1 antibody should detect a single band at approximately 17 kDa, corresponding to the observed molecular weight of histone H3 .

How should H3R26me1 antibodies be used in ChIP and ChIP-seq experiments?

While the search results don't provide a specific ChIP protocol for H3R26me1 antibodies, they contain information about ChIP considerations for histone PTM antibodies. Based on this, an optimized protocol would include:

Chromatin Preparation:

• Crosslink cells with 1% formaldehyde for 10 minutes at room temperature

• Quench with glycine (125 mM final concentration)

• Lyse cells and isolate nuclei

• Sonicate chromatin to fragments of 200-500 bp

• Check fragmentation by agarose gel electrophoresis

Immunoprecipitation:

• Pre-clear chromatin with protein A/G beads

• Set aside 5-10% of material as input control

• Incubate chromatin with H3R26me1 antibody:

  • Typically 3-5 μg per IP reaction

  • Include IgG control from the same species

• Incubate overnight at 4°C with rotation

• Add protein A/G beads and incubate for 2-4 hours

• Wash beads thoroughly (typically with low salt, high salt, LiCl, and TE buffers)

DNA Recovery:

• Elute chromatin from beads

• Reverse crosslinks (typically 65°C overnight)

• Treat with RNase A and Proteinase K

• Purify DNA using column-based or phenol-chloroform extraction

Analysis:

• For ChIP-qPCR: Design primers for regions of interest

• For ChIP-seq: Prepare libraries and sequence

• Include input normalization in all analyses

Important Considerations:

• Native vs. crosslinking conditions can affect antibody performance: "H3K79me2 Ab1, but not Ab2, modestly enriched for semi-synthetic nucleosomes marked with this PTM under native conditions, and neither antibody enriched for H3K79me2 nucleosomes under cross-linking conditions" .

• Include appropriate controls to assess specificity: "Analysis of wild-type mouse embryonic stem cells showed a strong correlation of read distribution between the two antibodies... To assess the specificity of these antibodies for H3K27 methylation, parallel ChIP-Seq experiments were performed in ES cells lacking H3K27 methylation" .

• Consider using internal spike-in controls: "We therefore sought to define more directly the specificity of histone PTM antibodies for endogenous nucleosomal epitopes by performing IPs of mouse ES cells (E14) spiked with a library of semi-synthetic DNA-barcoded mononucleosomes" .

What are the best practices for immunofluorescence with H3R26me1 antibodies?

Based on manufacturer recommendations and general principles for histone immunofluorescence:

Sample Preparation:

• Culture cells on glass coverslips or prepare tissue sections

• Fix samples:

  • For cells: 4% paraformaldehyde (10 minutes) or ice-cold methanol (10 minutes)

  • For tissues: Use appropriate fixation for the tissue type

Immunostaining Protocol:

• Permeabilize with 0.1-0.5% Triton X-100 in PBS (10 minutes)

• Block with 1-5% BSA or 0.2% fish scale gelatin in PBS (30-60 minutes)

• Incubate with primary H3R26me1 antibody at recommended dilution (1:50-1:200)

• Incubate overnight at 4°C or 1-2 hours at room temperature

• Wash 3 times with PBS

• Incubate with fluorophore-conjugated secondary antibody (1:200-1:1000)

• Counterstain nucleus with DAPI

• Mount using anti-fade mounting medium

Controls to Include:

• Secondary antibody-only control

• Peptide competition control

• Positive control (cell type known to express H3R26me1)

• Co-staining with total H3 antibody in a separate channel

Optimization Considerations:

• Test different fixation methods, as some epitopes are sensitive to fixation

• Adjust antibody concentration if signal is too weak or background is too high

• Consider antigen retrieval for tissue sections

• For co-staining, ensure primary antibodies are from different host species

An example of successful histone H3 IF is provided in search result : "ab1791 staining Histone H3 in HeLa by ICC/IF. Cells were fixed with methanol and blocked with 0.2% fish scale gelatin for 1 hour at 25°C. Samples were incubated with the primary antibody (1/300 in PBS + 0.2% gelatin) for 20 minutes at 25°C."

How can peptide competition assays validate H3R26me1 antibody specificity?

Peptide competition assays are valuable for validating antibody specificity. Based on the search results, an effective assay should include:

Peptide Selection:

• Target peptide: H3 peptide containing R26me1

• Negative control peptides:

  • Unmodified H3 peptide (same sequence, no methylation)

  • H3 peptides with other R26 modifications (R26me2a, R26me2s)

  • H3 peptides with methylation at other arginine residues

  • Peptides with combinations of modifications to test neighboring effects

Competition Protocol:

• Prepare antibody solutions:

  • Control: antibody alone in binding buffer

  • Test samples: antibody pre-incubated with excess competing peptide (5-10 μg peptide per 1 μg antibody)

• Incubate for 1-2 hours at room temperature or overnight at 4°C

• Use the pre-incubated antibody mixtures in parallel for your application (Western blot, IF, or ChIP)

Analysis and Interpretation:

• A specific antibody should show significant signal reduction when pre-incubated with the target H3R26me1 peptide

• Little or no signal reduction should be observed with unrelated peptides

• Partial signal reduction with similar modifications may indicate cross-reactivity

• Quantify the degree of competition (percent signal reduction) for each peptide

Search result describes a similar approach: "Additionally, peptide competition assays verified the interaction between the H3K14 antibodies and the H3K36ac peptide."

This methodology helps determine both the specificity for the R26me1 modification and potential cross-reactivity with similar epitopes, which is critical for accurate interpretation of experimental results.

What quantitative methods can assess H3R26me1 levels in cells and tissues?

Several approaches can be used to quantitatively assess H3R26me1 levels:

1. Western Blot Quantification:

• Extract histones using standardized protocols

• Load equal amounts of histone protein

• Include standard curves of synthetic H3R26me1 peptides

• Use fluorescent secondary antibodies for wider linear range

• Normalize H3R26me1 signal to total H3 signal

• Analyze using densitometry software

2. ELISA-Based Approaches:

• Commercial H3R26me1 antibodies are validated for ELISA applications

• Develop sandwich ELISA using anti-H3 capture and anti-H3R26me1 detection

• Include standard curves with synthetic peptides

• High-throughput compatible for multiple sample analysis

3. Immunofluorescence Quantification:

• Standardize image acquisition parameters

• Use automated image analysis algorithms

• Measure nuclear intensity of H3R26me1 staining

• Normalize to DAPI or total H3 staining

• Perform single-cell analysis of modification levels

4. Mass Spectrometry:

• Provides absolute quantification without antibody bias

• Use synthetic isotope-labeled peptides as internal standards

• Multiple Reaction Monitoring (MRM) for targeted analysis

• Enables detection of combinatorial modifications

5. ChIP-seq Quantification:

• Assess genome-wide distribution of H3R26me1

• Quantify peak numbers, intensities, and genomic distribution

• Compare enrichment at specific genomic features

• Integrate with gene expression data

6. Histone Methyltransferase Activity Assays:

• Similar to the approach described in search result : "We first evaluated our recombinant antibody and commercial antibodies for their ability to discriminate H3K9me2 and H3K9me3... This assay with 309M3-A clearly detected SUV39H1 activity and its inhibition by chaetocin"

• Develop in vitro assays to measure enzyme activity using H3R26me1 antibodies

Each method has advantages and limitations, and combining multiple approaches provides the most comprehensive assessment of H3R26me1 levels in biological samples.