HIST1H2AG (Ab-5) Antibody

What is HIST1H2AG (Ab-5) Antibody and what is its target?

HIST1H2AG (Ab-5) Antibody is a polyclonal antibody raised in rabbits against a peptide sequence around the lysine 5 (Lys-5) site derived from Human Histone H2A type 1. This antibody specifically targets histone H2A, which is a core component of nucleosomes . Histone H2A plays a central role in DNA packaging by helping to wrap and compact DNA into chromatin, thus regulating DNA accessibility to cellular machinery that requires DNA as a template . The antibody is designed to recognize post-translational modifications at or near the lysine 5 position, which is important for understanding epigenetic regulation mechanisms.

Which applications has HIST1H2AG (Ab-5) Antibody been validated for?

Based on current validation data, HIST1H2AG (Ab-5) Antibody has been successfully tested and validated for multiple laboratory applications:

*Validation may vary by catalog number and manufacturer .

What is the biological significance of histone H2A in epigenetic research?

Histone H2A is fundamental to epigenetic research as it is a key component of the nucleosome core, which forms the basic unit of chromatin structure. The protein plays crucial roles in:

• Chromatin compaction and DNA accessibility regulation

• Transcriptional control through modifications at specific residues

• DNA repair mechanisms and genomic stability

• Chromosome segregation during cell division

Importantly, post-translational modifications of histones, including H2A, constitute part of the "histone code" that regulates gene expression patterns. These modifications include acetylation, methylation, phosphorylation, and ubiquitination, all contributing to a complex regulatory network that influences cellular processes from development to disease progression .

How can HIST1H2AG (Ab-5) Antibody be used to investigate histone modifications in disease models?

HIST1H2AG (Ab-5) Antibody can be utilized in multiple advanced experimental approaches to investigate histone modifications in disease models:

• ChIP-Seq Analysis: The antibody can be employed in chromatin immunoprecipitation followed by sequencing to map genome-wide binding patterns of modified histone H2A, particularly at the lysine 5 position. This approach allows researchers to correlate specific histone modifications with gene expression changes in disease states.

• Multiplexed Immunofluorescence: By combining HIST1H2AG (Ab-5) with antibodies targeting other epigenetic markers, researchers can visualize the spatial relationships between different histone modifications in tissue samples from disease models.

• Single-Cell Epigenomic Profiling: Using HIST1H2AG (Ab-5) in flow cytometry-based applications allows for the assessment of histone modifications at the single-cell level, revealing heterogeneity in epigenetic states within complex tissues .

For optimal results in these advanced applications, validation in the specific experimental system is crucial before proceeding with large-scale studies.

What is the connection between HIST1H2AG antibody research and HIV-1 studies?

Intriguingly, research has revealed an unexpected connection between histone H2A-reactive B cells and HIV-1 neutralization. Studies have shown that:

• H2A-reactive IgM monoclonal antibodies display both autoreactivity and polyreactivity with self and foreign antigens

• These antibodies can neutralize multiple clades of tier 2 HIV-1

• In healthy individuals, H2A-reactive B cells are functionally anergic (non-responsive) due to peripheral tolerance mechanisms

• These B cells show increased expression of inhibitory mediators like CD5 and PTEN phosphatase

• They fail to mobilize calcium upon immunoreceptor stimulation, a characteristic marker of anergy

This research suggests that breaking tolerance in H2A-reactive B cells, such as through Toll-like receptor stimulation or CD4 T cell help, could potentially unlock a novel source of HIV-1 neutralizing antibodies . This represents an innovative intersection between autoimmunity research and infectious disease that could be further explored using HIST1H2AG (Ab-5) Antibody.

How does the specificity of HIST1H2AG (Ab-5) Antibody compare with other histone H2A antibodies?

The specificity of HIST1H2AG (Ab-5) Antibody is determined by its recognition of the peptide sequence around the lysine 5 position of Histone H2A type 1. This specificity offers several advantages in research:

When comparing with other H2A antibodies, researchers should consider:

• Whether they need an antibody specific to a particular modification (e.g., acetylation, methylation)

• Whether they need isoform-specific detection

• Whether they require a monoclonal or polyclonal antibody based on their experimental goals

What are the optimal sample preparation protocols for Western blot using HIST1H2AG (Ab-5) Antibody?

For optimal Western blot results with HIST1H2AG (Ab-5) Antibody, the following sample preparation protocol is recommended:

• Histone Extraction:

  • For cell culture: Use acid extraction methods (0.2N HCl or commercially available histone extraction kits)

  • For tissue samples: Homogenize tissues in extraction buffer containing histone deacetylase inhibitors and protease inhibitors

• Sample Preparation:

  • Use specialized histone-optimized loading buffers

  • Load 5-15 μg of acid-extracted histones per lane

  • Use 15-18% SDS-PAGE gels for optimal histone separation

  • Transfer to PVDF membrane (preferred over nitrocellulose for histones)

• Blotting Conditions:

  • Blocking: 5% non-fat dry milk or BSA in TBST, 1 hour at room temperature

  • Primary antibody incubation: Dilute HIST1H2AG (Ab-5) Antibody at 1:500-5000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

  • Wash 3-5 times with TBST

  • Secondary antibody: Anti-rabbit HRP-conjugated at 1:5000-10000

  • Detection: ECL substrate appropriate for expected signal strength

Note: Acid extraction of histones is critical for enriching histone content, as demonstrated in successful Western blot analysis using K562 cell acid extracts .

What controls should be included when using HIST1H2AG (Ab-5) Antibody for immunofluorescence?

When performing immunofluorescence experiments with HIST1H2AG (Ab-5) Antibody, include the following controls to ensure valid and interpretable results:

• Positive Control:

  • HeLa cells, which have been validated to express the target

  • Other human cell lines with known histone H2A expression patterns

• Negative Controls:

  • Primary antibody omission control (secondary antibody only)

  • Isotype control (rabbit IgG at the same concentration)

  • Peptide competition assay (pre-incubating antibody with the immunizing peptide)

• Specificity Controls:

  • Cells treated with histone deacetylase inhibitors (if studying acetylation)

  • Knockout or knockdown controls where available

  • Dual staining with another validated histone H2A antibody

• Technical Controls:

  • Autofluorescence control (no antibody)

  • Fixation control (compare different fixation methods; paraformaldehyde is typically recommended)

For optimal immunofluorescence results, use the recommended dilution of 1:50-200 and include DAPI or another nuclear stain to confirm nuclear localization .

How should HIST1H2AG (Ab-5) Antibody be stored and handled to maintain optimal activity?

Proper storage and handling of HIST1H2AG (Ab-5) Antibody is crucial for maintaining its functionality and specificity:

Handling Guidelines:

• Upon receipt, prepare small working aliquots to minimize freeze-thaw cycles

• Store in buffer containing 50% glycerol to prevent freezing damage

• Thaw aliquots completely before use and mix gently

• Avoid vortexing to prevent antibody denaturation

• Centrifuge briefly before opening tubes after thawing

• Keep on ice when preparing dilutions for experiments

• Note the presence of preservatives (0.03% Proclin 300) in the storage buffer

Following these storage recommendations will help ensure the antibody maintains its specified 12-month shelf life from the date of receipt .

What are common causes of nonspecific binding when using HIST1H2AG (Ab-5) Antibody and how can they be addressed?

Nonspecific binding is a common challenge when working with histone antibodies. For HIST1H2AG (Ab-5) Antibody, consider these troubleshooting approaches:

• High Background in Western Blots:

  • Increase blocking time/concentration (try 5% BSA instead of milk)

  • Increase washing steps (5× 5 minutes with TBST)

  • Optimize antibody dilution (test titration series from 1:500 to 1:5000)

  • Use more stringent washing buffers (increase Tween-20 concentration to 0.1-0.2%)

  • Pre-adsorb antibody with acetone powder from non-target species

• Nonspecific Nuclear Staining in Immunofluorescence:

  • Optimize fixation conditions (try 2-4% paraformaldehyde for 10 minutes)

  • Include permeabilization step (0.1-0.5% Triton X-100)

  • Include blocking step with normal serum from the same species as the secondary antibody

  • Reduce primary antibody concentration (test 1:100-1:200 dilutions)

  • Increase washing steps between antibody incubations

• Cross-reactivity with Other Histone Variants:

  • Perform peptide competition assays to confirm specificity

  • Include proper controls (histone H2A knockdown where possible)

  • Consider using more specific monoclonal antibodies for certain applications

  • When interpreting results, be aware of potential cross-reactivity with H2A variants

How can the sensitivity of detection be improved when working with low abundance histone modifications?

When studying low abundance histone modifications using HIST1H2AG (Ab-5) Antibody, consider these sensitivity-enhancing strategies:

• Sample Enrichment Techniques:

  • Use histone extraction kits specifically designed to preserve post-translational modifications

  • Implement chromatin immunoprecipitation (ChIP) to enrich for specific modified histones

  • Consider acid extraction methods that efficiently isolate histones from other cellular proteins

• Signal Amplification Methods:

  • For immunofluorescence: Use tyramide signal amplification (TSA)

  • For Western blots: Use high-sensitivity ECL substrates

  • Consider biotin-streptavidin amplification systems for enhanced detection

• Instrumentation Optimization:

  • For microscopy: Use high-sensitivity cameras and appropriate filter sets

  • For Western blot: Use longer exposure times and high-sensitivity imaging systems

  • For flow cytometry: Optimize voltage settings and compensation

• Protocol Refinements:

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize antibody concentration through careful titration

  • Reduce washing stringency slightly without compromising specificity

  • Use signal enhancers suitable for your detection system

How should experimental results be validated when studying histone H2A modifications with this antibody?

Validation of experimental results when studying histone H2A modifications requires a multi-faceted approach:

• Orthogonal Method Validation:

  • Confirm findings using multiple techniques (e.g., if detected by Western blot, validate with immunofluorescence)

  • Use mass spectrometry-based approaches to independently verify histone modifications

  • Employ ChIP-seq or similar techniques to correlate modification patterns with functional outcomes

• Genetic Validation:

  • Use CRISPR/Cas9-mediated modification of the target lysine residue

  • Employ histone modifying enzyme knockouts/knockdowns to alter modification states

  • Use cell lines with known histone modification profiles as benchmarks

• Pharmacological Validation:

  • Use histone deacetylase inhibitors (if studying acetylation)

  • Apply histone methyltransferase inhibitors (if studying methylation)

  • Include appropriate positive control treatments that induce the specific modification

• Antibody Validation:

  • Perform peptide competition assays with both modified and unmodified peptides

  • Use alternative antibodies targeting the same modification from different vendors

  • Include specificity controls as mentioned in section 3.2