Formyl-HIST1H2BC (K5) Antibody

What is the Formyl-HIST1H2BC (K5) Antibody and what biological modification does it detect?

The Formyl-HIST1H2BC (K5) Antibody is a research reagent designed to specifically recognize and bind to histone H2B proteins that contain a formyl group attached to the lysine residue at position 5. Histone H2B is one of the core components of nucleosomes, which wrap and compact DNA into chromatin. The formylation of lysine 5 represents a specific post-translational modification that may play important roles in regulating chromatin structure, DNA accessibility, and gene expression patterns .

Unlike other modifications such as butyrylation at the same position (K5), formylation has distinct biological implications and requires specific antibodies for accurate detection. Histones are subject to numerous post-translational modifications that collectively form the "histone code," which regulates DNA-templated processes including transcription, replication, and DNA repair .

How does Formyl-HIST1H2BC (K5) differ from other histone H2B modifications?

Formyl-HIST1H2BC (K5) represents a specific modification that differs from other well-characterized modifications at the same position, such as:

• Compared to Butyrylation (K5): While butyrylation at K5 involves the addition of a butyrate group (a short-chain fatty acid derivative), formylation involves the addition of a formyl group. These distinct chemical modifications likely recruit different effector proteins and serve unique biological functions .

• Compared to Acetylation: H2B K5 acetylation is generally associated with active transcription, whereas the biological function of H2B K5 formylation remains less well characterized but may have distinct regulatory roles in chromatin dynamics .

• Compared to Modifications at Other Positions: Unlike modifications at other H2B positions (such as K108 formylation), the K5 position is located at the N-terminus of the protein, making it more accessible and potentially more dynamic in its regulatory capacity .

The specificity of antibodies targeting these different modifications is critical for accurate experimental interpretation, as cross-reactivity between modification-specific antibodies can lead to misinterpretation of results .

What are the validated applications for Formyl-HIST1H2BC (K5) Antibody?

Based on current validation data, the Formyl-HIST1H2BC (K5) Antibody has been successfully employed in the following applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): Useful for quantitative detection of formylated H2B K5 in cell or tissue lysates .

• Immunocytochemistry (ICC): Enables visualization of the nuclear distribution of formylated H2B K5 in fixed cells .

• Western Blotting (WB): While specific protocols may need optimization, similar histone modification antibodies have been validated for western blot applications at dilutions ranging from 1:100 to 1:1000 .

• Chromatin Immunoprecipitation (ChIP): Though not explicitly validated for the formyl modification, similar histone antibodies have been successfully used in ChIP experiments to map genomic distribution of modifications .

The optimal experimental conditions, including antibody dilution, buffer composition, and incubation parameters, should be determined empirically for each specific application and experimental context.

What is the recommended protocol for immunohistochemistry with Formyl-HIST1H2BC (K5) Antibody?

For optimal results in immunohistochemistry with paraffin-embedded tissues (IHC-P), the following methodological approach is recommended:

• Sample Preparation:

  • Fix tissue samples in 10% neutral buffered formalin

  • Embed in paraffin and section to 4-6 μm thickness

  • Mount sections on positively charged slides

• Antigen Retrieval:

  • Deparaffinize sections in xylene and rehydrate through graded ethanol

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes

  • Allow slides to cool to room temperature

• Antibody Incubation:

  • Block endogenous peroxidase activity with 3% H₂O₂

  • Apply protein block to reduce non-specific binding

  • Incubate with Formyl-HIST1H2BC (K5) Antibody at a dilution of 1:50 to 1:200 (optimization recommended)

  • Incubate overnight at 4°C or for 1 hour at room temperature

• Detection and Visualization:

  • Apply appropriate secondary antibody (such as anti-rabbit IgG)

  • Develop signal using DAB or other suitable chromogen

  • Counterstain with hematoxylin, dehydrate, and mount

Positive controls should include human liver, mouse lung, or rat brain tissues, which have been validated for this application .

How can I validate the specificity of Formyl-HIST1H2BC (K5) Antibody for my experiments?

Validating antibody specificity is crucial for accurate interpretation of results. For Formyl-HIST1H2BC (K5) Antibody, consider these validation approaches:

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess synthetic peptide containing the formylated K5 modification

  • In parallel, perform your experiment with untreated antibody

  • Signal elimination in the peptide-competed condition confirms specificity

• Modification-Specific Controls:

  • Compare signal patterns between samples treated with or without histone deacetylase inhibitors or agents that affect formylation

  • For example, treatment with sodium butyrate (which influences histone modifications) should alter the signal pattern

• Peptide Array Analysis:

  • Test antibody recognition against a panel of differentially modified histone peptides

  • This approach, similar to that used in The Histone Antibody Specificity Database, can reveal potential cross-reactivity with other modifications

• Genetic Controls:

  • If possible, use cells with targeted mutations at H2B K5 that prevent formylation

  • Signal absence in these samples would confirm specificity

A comprehensive validation should include at least two independent approaches to establish confidence in antibody specificity.

Does the Formyl-HIST1H2BC (K5) Antibody cross-react with other histone modifications?

Cross-reactivity is a significant concern with histone modification antibodies. Based on available data and similar antibody behaviors:

• Potential Cross-Reactivity:

  • Histone modification antibodies often show recognition patterns dependent on adjacent modifications

  • As demonstrated with H4 acetyl antibodies, there may be enhanced binding to epitopes with multiple modifications

• Modification-Adjacent Effects:

  • The antibody specificity may be influenced by modifications at nearby residues

  • For example, modifications at adjacent lysines (K4, K8) could potentially affect recognition of K5 formylation

• Recommended Validation:

  • Test against synthetic peptides containing:

    • H2B K5 with different modifications (acetylation, butyrylation, methylation)

    • H2B with formylation at different lysine positions

    • H2B K5 formylation with additional modifications at adjacent residues

The peptide microarray approach, as utilized in The Histone Antibody Specificity Database, represents an effective method for comprehensive assessment of potential cross-reactivity .

How can Formyl-HIST1H2BC (K5) Antibody be effectively used in ChIP-seq experiments?

While specific ChIP-seq protocols for Formyl-HIST1H2BC (K5) Antibody may require optimization, the following methodological approach is recommended based on successful ChIP-seq experiments with similar histone modification antibodies:

• Chromatin Preparation:

  • Cross-link cells with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 125 mM glycine

  • Isolate nuclei and fragment chromatin to 200-500 bp using sonication

  • Verify fragmentation efficiency by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate 2-5 μg of Formyl-HIST1H2BC (K5) Antibody with 25-50 μg of chromatin overnight at 4°C

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

  • Perform stringent washing to remove non-specific interactions

• Library Preparation and Sequencing:

  • Reverse cross-links and purify DNA

  • Prepare sequencing libraries following standard protocols

  • Include appropriate controls (input DNA, IgG control)

• Data Analysis Considerations:

  • When analyzing ChIP-seq data, consider the genomic distribution of H2B K5 formylation

  • Compare with other histone marks to understand the regulatory context

  • Validate peaks by ChIP-qPCR at selected loci

To establish specificity, parallel ChIP-seq experiments can be performed in cells treated with agents that alter histone formylation patterns, similar to the approach used for validating H3K27 methylation antibodies in cells lacking EED .

What is the relationship between H2B K5 formylation and other epigenetic marks in the regulation of gene expression?

The interplay between H2B K5 formylation and other epigenetic modifications forms a complex regulatory network:

This multifaceted analysis would provide insights into the functional role of H2B K5 formylation within the histone code .

What are common challenges when using Formyl-HIST1H2BC (K5) Antibody and how can they be addressed?

Researchers may encounter several challenges when working with histone modification antibodies, including:

• High Background Signal:

  • Problem: Non-specific staining in immunohistochemistry or western blots

  • Solution: Increase blocking time/concentration, optimize antibody dilution (try 1:100 to 1:200), perform more stringent washes, or use a different blocking reagent

• Weak or No Signal:

  • Problem: Insufficient detection of the target modification

  • Solution: Optimize antigen retrieval conditions, increase antibody concentration, extend incubation time, or enhance signal with amplification systems

• Cross-Reactivity Issues:

  • Problem: Antibody recognizes unintended modifications

  • Solution: Validate specificity using peptide competition assays, include appropriate controls, or consider using a more specific antibody lot

• Inconsistent Results Between Experiments:

  • Problem: Variable signal intensity or patterns

  • Solution: Standardize sample preparation, maintain consistent antibody lot numbers, and include positive controls in each experiment

• ChIP Efficiency Problems:

  • Problem: Low yield in chromatin immunoprecipitation

  • Solution: Optimize chromatin fragmentation, increase antibody amount, extend incubation time, or modify washing conditions to improve specificity while maintaining yield

For all applications, empirical optimization is essential, as conditions may vary depending on sample type, fixation method, and experimental design .

How should I quantify and interpret Formyl-HIST1H2BC (K5) signals in different experimental contexts?

• Western Blot Quantification:

  • Normalize H2B K5 formylation signal to total H2B or another loading control

  • Use densitometry software for objective quantification

  • Present data as relative fold change compared to control conditions

• Immunofluorescence Analysis:

  • Measure nuclear intensity across multiple cells (n>50)

  • Report mean fluorescence intensity with appropriate statistical analysis

  • Consider subcellular distribution patterns, not just total signal intensity

• ChIP-qPCR Quantification:

  • Calculate percent input or fold enrichment over IgG control

  • Analyze multiple genomic regions, including positive and negative control loci

  • Present data with appropriate error bars and statistical significance

• Interpretation Considerations:

  • Changes in H2B K5 formylation should be interpreted in the context of:

    • Cell type and physiological state

    • Treatment conditions that might affect global histone modifications

    • Correlation with functional outcomes (gene expression, chromatin accessibility)

  • Consider the dynamic nature of histone modifications and potential turnover rates

Statistical analysis should be rigorous and appropriate for the experimental design, with clear reporting of sample sizes, replicates, and significance thresholds .

What is the biological function of H2B K5 formylation in chromatin regulation and cellular processes?

The biological significance of H2B K5 formylation is still being elucidated, but current understanding suggests several important roles:

• Chromatin Structure Regulation:

  • H2B is a core component of nucleosomes that wrap and compact DNA

  • Formylation at K5 likely affects the physical properties of chromatin, potentially influencing DNA accessibility to transcription factors and other regulatory proteins

• Transcriptional Regulation:

  • Based on studies of other histone modifications, H2B K5 formylation may play a role in:

    • Transcriptional activation or repression

    • Recruitment of specific protein complexes to chromatin

    • Modulation of other histone modifications through cross-talk mechanisms

• Cellular Defense Functions:

  • Histones, including H2B, have been reported to have antibacterial and antifungal properties

  • H2B may contribute to the formation of functional antimicrobial barriers in tissues such as the colonic epithelium

  • The formylation of K5 could potentially modulate these antimicrobial properties

• Cell Cycle Regulation:

  • Histone modifications often show cell cycle-dependent patterns

  • H2B K5 formylation may have specific roles during DNA replication, mitosis, or other cell cycle phases

• Response to Cellular Stress:

  • Histone modifications can change in response to environmental stressors

  • H2B K5 formylation may serve as a stress-responsive mark that helps cells adapt to changing conditions

Further research using specific antibodies against H2B K5 formylation will help elucidate its precise functions in different cellular contexts and disease states .

How does H2B K5 formylation compare across different cell types and disease states?

Understanding the variation in H2B K5 formylation patterns across biological contexts provides insights into its functional relevance:

• Tissue-Specific Patterns:

  • Different tissues exhibit varying levels of histone modifications

  • Immunohistochemical analysis reveals that H2B K5 formylation may be detected in human liver, mouse lung, and rat brain tissues, suggesting tissue-specific roles

  • Comprehensive tissue mapping could reveal cell types where this modification is particularly abundant

• Disease-Associated Changes:

  • Altered histone modifications have been implicated in various diseases, particularly cancer

  • Changes in H2B K5 formylation might correlate with:

    • Tumor progression and aggressiveness

    • Response to treatment

    • Patient prognosis

• Developmental Dynamics:

  • Histone modifications often change during development

  • Tracking H2B K5 formylation across developmental stages could reveal its role in cell differentiation and tissue specification

• Comparative Analysis Table:

• Environmental Responsiveness:

  • Environmental factors and cellular stressors may influence H2B K5 formylation patterns

  • Treatment with epigenetic modulators such as sodium butyrate can alter histone modification landscapes

These comparative analyses are essential for understanding the biological significance of H2B K5 formylation and its potential as a therapeutic target or biomarker in various diseases.