Glutaryl-HIST1H2BC (K120) Antibody

What is Glutaryl-HIST1H2BC (K120) Antibody and what role does it play in epigenetic research?

Glutaryl-HIST1H2BC (K120) Antibody is a high-quality polyclonal antibody specifically designed for detecting glutarylation at lysine 120 of the histone protein Hist1H2BC. Raised in rabbits, this antibody exhibits high sensitivity and specificity for human samples with this particular modification .

In epigenetic research, this antibody serves as a critical tool for investigating histone glutarylation, which is a post-translational modification (PTM) that can influence chromatin structure and function. Histone proteins, including Hist1H2BC, play essential roles in packaging DNA into chromatin and regulating gene expression . The ability to specifically detect glutarylation at lysine 120 allows researchers to study how this modification affects various cellular processes and contributes to epigenetic regulation.

How does histone glutarylation differ from other histone modifications in functional impact?

Histone glutarylation is one of many post-translational modifications that affect histone proteins, but it has distinct characteristics compared to more well-studied modifications like methylation and acetylation.

While acetylation typically neutralizes the positive charge of lysine residues and loosens chromatin structure, glutarylation adds a larger chemical group with a negative charge, potentially creating more significant structural changes to chromatin . Unlike methylation which can exist in multiple states (mono-, di-, or tri-methylation) with different functional outcomes, glutarylation is a single-state modification but may have context-dependent effects.

The functional impact of glutarylation at specific sites such as K120 on Hist1H2BC may influence transcriptional regulation, DNA repair processes, and potentially interact with other histone modifications in the broader "histone code" . Understanding these unique aspects of glutarylation helps researchers interpret experimental results in the proper context of epigenetic regulation.

What are the key differences between Glutaryl-HIST1H2BC antibodies targeting different lysine residues (K116 vs. K120)?

The search results reveal antibodies targeting glutarylation at different lysine residues on HIST1H2BC, specifically K116 and K120 . These antibodies differ in:

The biological significance of glutarylation at these distinct lysine residues may vary, potentially affecting different aspects of chromatin regulation or protein interactions. When designing experiments, researchers should select the appropriate antibody based on which specific modification site they aim to study .

What are the validated applications for Glutaryl-HIST1H2BC (K120) Antibody and how should they be optimized?

The Glutaryl-HIST1H2BC (K120) Antibody has been validated for several experimental applications:

• Western Blotting (WB): The antibody is validated for detecting glutarylated HIST1H2BC in protein samples, with recommended dilutions of 1:100-1:1000 .

• Enzyme-Linked Immunosorbent Assay (ELISA): The antibody can be used in ELISA applications with recommended dilutions of 1:2000-1:10000 .

For optimal results in Western blotting:

• Use fresh samples with protease inhibitors and deacetylase inhibitors

• Include appropriate controls (positive, negative, and loading controls)

• Optimize blocking conditions to minimize background

• Validate antibody specificity using peptide competition assays

• Consider enhanced chemiluminescence (ECL) detection for optimal sensitivity

For ELISA applications:

• Establish standard curves using known concentrations of glutarylated peptides

• Optimize coating buffer conditions and antibody concentrations

• Validate using appropriate controls

• Consider using streptavidin-HRP systems for detection enhancement

What is the recommended sample preparation protocol for detecting histone glutarylation?

For effective detection of histone glutarylation, careful sample preparation is essential:

• Histone Extraction:

  • Use dedicated histone extraction kits or acid extraction methods

  • Include deacetylase/deglutarylase inhibitors (e.g., sodium butyrate, nicotinamide)

  • Maintain cold temperatures throughout extraction to prevent modification loss

• Storage Conditions:

  • Store antibody at -20°C or -80°C to maintain activity

  • Avoid repeated freeze-thaw cycles

  • Prepare aliquots for single use

• Sample Handling:

  • Use freshly prepared samples when possible

  • For long-term storage, add glycerol (50%) and store at recommended temperature (-20°C/-80°C)

  • Include 0.03% Proclin 300 as a preservative in storage buffers

• Buffer Composition:

  • Use PBS buffer (0.01M, pH 7.4) with 50% glycerol for sample preparation

  • Ensure proper pH (7.4) for optimal antibody recognition of the epitope

How can ChIP experiments be optimized using Glutaryl-HIST1H2BC (K120) Antibody?

While the search results don't specifically mention ChIP validation for the K120 antibody, similar histone modification antibodies have been used in ChIP experiments . For optimizing ChIP with Glutaryl-HIST1H2BC (K120) Antibody:

• Crosslinking Optimization:

  • Test different formaldehyde concentrations (0.5-1.5%) and crosslinking times

  • Include glycine quenching to stop crosslinking reaction

• Sonication Parameters:

  • Optimize sonication conditions to generate fragments of 200-500bp

  • Verify fragment size by agarose gel electrophoresis

• Antibody Concentration:

  • Titrate antibody amounts (typically 2-10μg per ChIP reaction)

  • Include IgG controls matched to the host species (rabbit)

• Washing Stringency:

  • Test different washing buffers with increasing salt concentrations

  • Balance between reducing background and maintaining specific binding

• Validation Methods:

  • Use qPCR primers targeting regions known to be enriched for H2B modifications

  • Include positive controls (actively transcribed genes) and negative controls (silent genes)

How does glutarylation at K120 interact with other histone modifications in the broader epigenetic landscape?

Histone modifications often function in combination rather than isolation, creating a complex "histone code" that regulates chromatin structure and gene expression. The glutarylation at K120 of HIST1H2BC likely participates in this code through various interactions:

• Crosstalk Mechanisms:

  • Glutarylation may compete with other lysine modifications (acetylation, methylation, ubiquitination) at the same residue

  • Modifications on neighboring residues may influence enzyme accessibility for glutarylation/deglutarylation

  • Glutarylation may recruit or repel specific chromatin-modifying complexes

• Sequential Modifications:

  • Certain modifications may need to occur before glutarylation is possible

  • Glutarylation may serve as a prerequisite for subsequent modifications

• Functional Consequences:

  • The combination of glutarylation with other modifications may have synergistic or antagonistic effects on transcription

  • Different modification patterns may signal for distinct cellular processes (e.g., DNA repair, replication, transcription)

Understanding these interactions requires techniques like sequential ChIP, mass spectrometry, and combinatorial antibody approaches to map the co-occurrence of multiple modifications .

What controls should be included when studying histone glutarylation to ensure data reliability?

Robust experimental design for studying histone glutarylation requires multiple controls:

• Antibody Specificity Controls:

  • Peptide competition assays using glutarylated and unmodified peptides

  • Testing on samples with enzymatically removed glutarylation

  • Cross-reactivity testing with other acylated histones

• Sample Processing Controls:

  • Include deglutarylase inhibitors in all buffers

  • Process all experimental and control samples identically

  • Monitor for consistency in extraction efficiency

• Experimental Controls:

  • Positive controls: cell lines/tissues known to have high glutarylation levels

  • Negative controls: samples treated with deglutarylases

  • Treatment controls: metabolic interventions known to alter glutarylation (e.g., glutaryl-CoA dehydrogenase modulators)

• Technical Controls:

  • Loading controls for Western blots (total histone H3 or H2B)

  • Input controls for ChIP experiments

  • Standard curves for quantitative analyses

Including these controls helps distinguish true biological effects from technical artifacts and ensures reproducibility of findings across independent experiments .

What approaches can help resolve contradictory findings in histone glutarylation studies?

Resolving contradictions in histone glutarylation research requires systematic investigation:

• Antibody Validation:

  • Verify antibody specificity through multiple methods

  • Compare results with different antibody clones/sources

  • Validate findings with mass spectrometry when possible

• Methodological Standardization:

  • Document detailed protocols including buffer compositions

  • Standardize extraction and detection methods across studies

  • Report all experimental parameters that might influence results

• Biological Context Considerations:

  • Cell type/tissue specificity of glutarylation patterns

  • Temporal dynamics of the modification

  • Metabolic state of the system under study

• Integrated Approaches:

  • Combine antibody-based detection with mass spectrometry

  • Correlate functional outcomes with modification levels

  • Use genetic and pharmacological interventions to validate causal relationships

• Collaborative Validation:

  • Replicate key experiments in independent laboratories

  • Share reagents and protocols to ensure comparability

  • Establish consensus on technical standards

These approaches can help determine whether contradictions represent technical artifacts or genuine biological complexity in histone glutarylation patterns .

Why might I get inconsistent results with Glutaryl-HIST1H2BC (K120) Antibody and how can these issues be addressed?

Inconsistent results when using Glutaryl-HIST1H2BC (K120) Antibody can stem from several sources:

• Antibody-Related Issues:

  • Degradation due to improper storage or repeated freeze-thaw cycles

  • Lot-to-lot variability in polyclonal antibody preparations

  • Solution: Store antibody at -20°C or -80°C in small aliquots to avoid repeated freezing and thawing

• Sample Preparation Problems:

  • Loss of glutarylation during extraction due to active deglutarylases

  • Inconsistent extraction efficiency between experiments

  • Solution: Include deglutarylase inhibitors and standardize extraction protocols

• Technical Variations:

  • Inconsistent blocking conditions causing variable background

  • Differences in incubation times or temperatures

  • Solution: Develop detailed SOPs and maintain consistent conditions across experiments

• Biological Variability:

  • Cell cycle-dependent changes in glutarylation levels

  • Metabolic state influencing glutarylation dynamics

  • Solution: Synchronize cells when possible and control for metabolic conditions

• Detection System Limitations:

  • Variable sensitivity of detection reagents

  • Non-linear signal response

  • Solution: Include standard curves and validate the linear range of detection

Addressing these issues requires systematic troubleshooting and careful documentation of all experimental parameters.

How can I distinguish between specific and non-specific binding in glutarylation detection experiments?

Distinguishing specific from non-specific binding is crucial for accurate interpretation:

• Peptide Competition Assays:

  • Pre-incubate antibody with glutarylated peptide (specific competitor)

  • Pre-incubate with unmodified peptide (control)

  • Specific signal should be eliminated only by the glutarylated peptide

• Validation with Multiple Techniques:

  • Compare antibody-based detection with mass spectrometry

  • Use orthogonal approaches like in vitro glutarylation assays

  • Employ genetic approaches (e.g., mutating K120 to arginine)

• Controls for Western Blot:

  • Include samples with enzymatically removed glutarylation

  • Use cell lines with known glutarylation status

  • Test specificity with dot blots using modified and unmodified peptides

• Signal Validation Approaches:

  • Verify expected molecular weight (approximately 14-15kDa for H2B)

  • Confirm signal disappearance after appropriate treatments

  • Test across multiple cell types with expected glutarylation differences

These approaches collectively help ensure that observed signals genuinely represent glutarylation at K120 rather than artifacts or cross-reactivity .

What are the common pitfalls in quantifying histone glutarylation levels and how can they be overcome?

Quantifying histone glutarylation presents several challenges:

• Dynamic Range Limitations:

  • Western blot has limited dynamic range for quantification

  • Solution: Use digital imaging systems and validate linear range with standard curves

• Reference Standard Issues:

  • Lack of universally accepted standards for glutarylation levels

  • Solution: Develop internal standards and report relative changes rather than absolute values

• Normalization Challenges:

  • Variations in total histone loading

  • Solution: Normalize to total H2B or total protein, and verify equal loading with multiple controls

• Technical Variability:

  • Batch effects in sample processing

  • Solution: Process all experimental conditions simultaneously when possible

• Antibody Saturation:

  • Non-linear response at high modification levels

  • Solution: Perform antibody titrations and ensure measurements are within the linear range

• Extraction Efficiency Variability:

  • Inconsistent recovery of modified histones

  • Solution: Use spike-in controls and validate extraction protocols

Overcoming these challenges requires rigorous method validation, appropriate controls, and careful consideration of quantification approaches .

How is Glutaryl-HIST1H2BC (K120) being applied in current epigenetic research on disease mechanisms?

Histone glutarylation research is expanding into disease-related contexts:

• Cancer Biology:

  • Investigating altered glutarylation patterns in various cancer types

  • Exploring connections between metabolic dysregulation and histone glutarylation

  • Examining potential of glutarylation as biomarkers for cancer progression

• Neurodegenerative Disorders:

  • Studying glutarylation changes in conditions like Alzheimer's and Parkinson's

  • Investigating glutarylation in models of neuronal stress and aging

  • Exploring links between glutaryl-CoA metabolism and neurodegeneration

• Metabolic Diseases:

  • Examining how metabolic disorders affect histone glutarylation patterns

  • Studying the interplay between diet, metabolism, and epigenetic modifications

  • Investigating glutarylation as a link between metabolism and gene regulation

Understanding histone glutarylation in these contexts may reveal new insights into disease mechanisms and potentially identify novel therapeutic targets .

What technological advances are enhancing the study of site-specific histone glutarylation?

Several technological developments are advancing histone glutarylation research:

• Mass Spectrometry Innovations:

  • Improved sensitivity for detecting low-abundance modifications

  • Quantitative approaches like SILAC and TMT labeling

  • Top-down proteomics for analyzing intact histone proteins

• Genomic Mapping Technologies:

  • ChIP-seq adaptations for mapping glutarylation genome-wide

  • CUT&RUN and CUT&Tag methods for improved resolution

  • Single-cell approaches for heterogeneity analysis

• Synthetic Biology Tools:

  • Engineered deglutarylases and glutaryltransferases

  • Site-specific incorporation of glutarylated lysines using genetic code expansion

  • CRISPR-based epigenetic editors targeting glutarylation machinery

• Computational Approaches:

  • Machine learning algorithms for predicting glutarylation sites

  • Integrative analysis of multiple histone modifications

  • Molecular dynamics simulations of glutarylation effects on chromatin structure

These technological advances collectively enable more comprehensive and precise investigation of histone glutarylation and its biological roles .

How does glutarylation compare to other acylation modifications in terms of regulatory mechanisms and functional impact?

Histone acylation modifications form a diverse family with distinct properties:

Glutarylation is distinctive due to its larger size and negative charge, which may create more significant structural alterations to chromatin compared to other acylations. The enzymatic machinery regulating glutarylation is still being characterized, with sirtuins likely playing important roles.

The functional impacts of glutarylation appear context-dependent, potentially influenced by:

• Local chromatin environment

• Co-occurring modifications

• Metabolic state of the cell

• Tissue-specific regulatory factors