Hydroxyl-Histone H2A type 1-B/E (Y39) Recombinant Monoclonal Antibody

What is Hydroxyl-Histone H2A type 1-B/E (Y39) and why is it significant?

Hydroxyl-Histone H2A type 1-B/E (Y39) refers to histone H2A that is hydroxylated specifically at the tyrosine 39 residue. Histone H2A is one of the four core histones (H2A, H2B, H3, and H4) that form the nucleosome, the basic structural unit of chromatin. Each nucleosome consists of approximately 146 base pairs of DNA wrapped around a histone octamer composed of pairs of these four core histones .

The hydroxylation at Y39 is a specific post-translational modification that likely plays a role in regulating chromatin structure and function. Histones and their modifications are central to transcription regulation, DNA repair, DNA replication, and chromosomal stability . The specific hydroxylation at Y39 may alter nucleosome dynamics and influence DNA accessibility to cellular machinery that requires DNA as a template.

How does this recombinant monoclonal antibody differ from polyclonal antibodies?

This recombinant monoclonal antibody is produced using recombinant technology, typically expressed in systems such as HEK293F cells . Unlike polyclonal antibodies, which are derived from multiple B cell lineages and recognize various epitopes, this monoclonal antibody:

• Derives from a single B cell clone, ensuring consistent recognition of the same epitope

• Offers higher specificity as it detects only the hydroxylation at Y39 of histone H2A

• Provides better reproducibility between experiments and batches

• Can be produced with consistent quality through recombinant technology without animal immunization

• Has been validated to detect endogenous levels of Histone H2A protein only when hydroxylated at Tyr39

The recombinant nature ensures that the antibody has defined properties and constant affinity for the target, making experimental results more reliable and reproducible across different research settings.

What are the validated applications for this antibody?

The Hydroxyl-Histone H2A-Y39 recombinant monoclonal antibody has been validated for several applications:

When using this antibody for Western blot analysis, it has successfully detected hydroxylated H2A-Y39 in lysates from multiple cell lines, including HeLa (human) and NIH/3T3 (mouse) cells .

How should I optimize Western blot protocols for this antibody?

For optimal Western blot results with the Hydroxyl-Histone H2A-Y39 antibody, consider these methodological guidelines:

• Sample preparation:

  • Use fresh cell or tissue lysates whenever possible

  • Include protease and phosphatase inhibitors in lysis buffers to preserve post-translational modifications

  • For histone extraction, consider using specialized histone extraction protocols or commercial kits

• Loading and separation:

  • Load approximately 10-25 μg of total protein per lane

  • Use 15% or gradient SDS-PAGE gels for better separation of low molecular weight histones (H2A has a calculated MW of 14 kDa, observed at ~17 kDa)

• Transfer and blocking:

  • Use PVDF membrane for optimal protein binding

  • For blocking, 5% BSA in TBST is recommended over milk , as milk can contain phosphatases that might affect phosphorylated proteins

• Antibody incubation:

  • Primary antibody dilution: 1:500 - 1:2000 in 5% BSA/TBST

  • Incubate overnight at 4°C for optimal results

  • Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:10000 dilution

• Detection:

  • Use enhanced chemiluminescence (ECL) detection systems

  • Exposure times may need optimization depending on expression levels

Evidence from validation studies shows successful detection of hydroxylated H2A-Y39 in multiple cell lines using these parameters .

What considerations should be taken for immunohistochemistry with this antibody?

When performing immunohistochemistry with this antibody, the following methodological considerations are important:

• Tissue preparation:

  • Paraffin-embedded sections should be properly fixed (typically with 10% neutral buffered formalin)

  • Perform antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Section thickness of 4-6 μm is generally optimal

• Antibody application:

  • Recommended dilution: 1:50 - 1:200

  • Incubation time: 1-2 hours at room temperature or overnight at 4°C

  • Use a humidity chamber to prevent tissue drying

• Detection system:

  • Use a polymer-based detection system for enhanced sensitivity

  • DAB (3,3'-diaminobenzidine) is commonly used as the chromogen

  • Counterstain with hematoxylin for nuclear visualization

• Validated tissues:

  • The antibody has been successfully used on human colon cancer tissue

  • Also validated on human tonsil, mouse colon, and mouse testis tissues

  • Counter staining with hematoxylin provides good nuclear contrast

Immunohistochemical analysis reveals nuclear localization patterns consistent with the chromatin-associated nature of histone H2A, with variations in staining intensity potentially indicating differences in hydroxylation levels across cell types and tissues .

What are common issues when working with histone antibodies and how can they be addressed?

Working with histone antibodies presents several technical challenges:

• Cross-reactivity with histone variants:

  • Problem: H2A has multiple variants that share sequence similarity

  • Solution: Verify the specificity of the antibody through peptide competition assays or using knockout/knockdown controls

• Post-translational modification (PTM) specificity:

  • Problem: Ensuring the antibody detects only Y39 hydroxylation and not other modifications

  • Solution: Use PTM-specific controls and validated antibody lots that have been tested for specificity against hydroxylated vs. non-hydroxylated peptides

• Extraction efficiency:

  • Problem: Histones are tightly bound to DNA in chromatin structures

  • Solution: Use specialized histone extraction protocols that include high salt concentration or acid extraction methods

• Signal-to-noise ratio:

  • Problem: Background staining can obscure specific signals

  • Solution: Optimize blocking conditions (5% BSA is recommended ), increase antibody dilution, or use alternative blocking reagents

• Storage and stability issues:

  • Problem: Antibody activity loss over time

  • Solution: Store at -20°C and avoid freeze/thaw cycles , aliquot upon receipt for long-term use

Researchers have successfully addressed these challenges through careful protocol optimization and proper controls, as evidenced by the clean Western blot results shown in the validation data .

How can I validate the specificity of this antibody for my experiments?

Validating antibody specificity is crucial for reliable results. For Hydroxyl-Histone H2A-Y39 antibody, consider these validation approaches:

• Peptide competition assay:

  • Pre-incubate the antibody with hydroxylated H2A-Y39 peptide before application

  • A specific antibody will show reduced or eliminated signal

• Comparison with non-hydroxylated controls:

  • Test samples with known hydroxylation status

  • Compare with samples treated with tyrosine phosphatase to remove hydroxyl groups

• Knockout/knockdown validation:

  • Use CRISPR/Cas9 to generate Y39F mutants (phenylalanine cannot be hydroxylated)

  • Compare signal between wild-type and mutant samples

• Multi-antibody approach:

  • Use multiple antibodies targeting the same modification from different vendors

  • Concordant results increase confidence in specificity

• Mass spectrometry correlation:

  • Confirm hydroxylation status using mass spectrometry

  • Correlate antibody-based detection with MS results

The antibody has been validated to detect endogenous levels of Histone H2A protein only when hydroxylated at tyrosine 39, with validation shown across multiple species including human, mouse, and rat .

What is the biological significance of histone H2A Y39 hydroxylation?

Histone H2A hydroxylation at Y39 represents an important post-translational modification with several biological implications:

• Chromatin structure regulation:

  • Hydroxylation likely alters nucleosome stability and DNA-histone interactions

  • May influence higher-order chromatin structures through changes in nucleosome dynamics

• Transcriptional regulation:

  • Post-translational modifications of histones constitute the "histone code" that regulates gene expression

  • Hydroxylation at Y39 likely affects DNA accessibility to transcription machinery

• DNA damage response:

  • Histones play central roles in DNA repair processes

  • Hydroxylation may serve as a signal for recruitment of DNA repair factors

• Cell cycle regulation:

  • Histone modifications change throughout the cell cycle

  • Y39 hydroxylation patterns may correlate with specific cell cycle phases

• Epigenetic inheritance:

  • Stable histone modifications contribute to epigenetic memory

  • Understanding hydroxylation dynamics helps elucidate epigenetic mechanisms

While the specific molecular mechanisms by which Y39 hydroxylation affects these processes requires further research, studies using the Hydroxyl-Histone H2A-Y39 antibody have begun to reveal tissue-specific patterns in human and mouse samples , suggesting context-dependent biological roles.

How does hydroxylation of H2A-Y39 integrate with other histone modifications?

Histone modifications rarely function in isolation. The hydroxylation of H2A-Y39 likely interacts with other modifications in several ways:

• Modification crosstalk:

  • Hydroxylation at Y39 may influence or be influenced by nearby modifications

  • For example, acetylation of neighboring lysine residues or methylation of adjacent amino acids

• Combinatorial effects:

  • The specific combination of modifications (including Y39 hydroxylation) creates a "code" read by effector proteins

  • This code determines the functional outcome on chromatin structure and gene expression

• Modification dynamics:

  • Histone modifications are dynamic, with enzymes adding and removing specific modifications

  • Hydroxylation may exhibit temporal patterns related to other modifications during cellular processes

• Reader proteins:

  • Specific proteins recognize hydroxylated Y39 and mediate downstream effects

  • These readers may also interact with proteins recognizing other modifications

• Evolutionary conservation:

  • The high conservation of H2A across species suggests functional importance of this modification

  • The antibody's cross-reactivity with human, mouse, and rat samples indicates conservation of this modification site

Research using this antibody across different experimental systems can help elucidate these complex interactions and their biological significance in different cellular contexts.

What cell types and tissues show significant levels of H2A-Y39 hydroxylation?

Based on immunohistochemistry data from validation studies, H2A-Y39 hydroxylation has been detected in several tissues and cell types:

The nuclear localization observed in these studies is consistent with the role of histones in chromatin structure. The variation in staining intensity across different tissues suggests tissue-specific regulation of this modification. The presence of hydroxylated H2A-Y39 in both normal and cancer tissues indicates its fundamental role in cellular processes, though potential differences in modification levels between normal and pathological states warrant further investigation.

What are the optimal storage and handling conditions for this antibody?

For maximum stability and retention of activity, the Hydroxyl-Histone H2A-Y39 antibody should be handled according to these guidelines:

• Storage temperature:

  • Store at -20°C for long-term stability

  • Avoid storage at 4°C for extended periods

• Buffer composition:

  • Typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • This formulation helps maintain antibody stability during freeze/thaw cycles

• Aliquoting:

  • Upon receipt, divide into small aliquots to minimize freeze/thaw cycles

  • Each freeze/thaw cycle can reduce antibody activity

• Working solution:

  • Dilute only the amount needed for immediate use

  • Return stock solution to -20°C immediately after use

• Shipping and temporary storage:

  • Can temporarily be stored at 4°C upon receipt

  • For shipping, wet ice conditions are appropriate

• Expiration:

  • Check manufacturer's recommended expiration date

  • Proper storage can extend useful shelf life

Following these guidelines ensures optimal antibody performance across multiple applications and experimental conditions.

What controls should be included when using this antibody?

Including appropriate controls is essential for interpreting results with the Hydroxyl-Histone H2A-Y39 antibody:

• Positive controls:

  • Cell lines with known H2A-Y39 hydroxylation (e.g., HeLa, NIH/3T3)

  • Tissues with confirmed expression (e.g., human colon cancer tissue)

• Negative controls:

  • Primary antibody omission to assess secondary antibody specificity

  • Samples treated with tyrosine phosphatase to remove hydroxyl groups

  • Y39F mutant cells (if available) where hydroxylation cannot occur

• Loading controls:

  • For Western blots, total H2A antibody to normalize for histone content

  • Housekeeping proteins for total protein normalization

• Peptide competition:

  • Pre-incubation with hydroxylated Y39 peptide should abolish specific signal

  • Pre-incubation with non-hydroxylated peptide should not affect signal

• Cross-reactivity controls:

  • Test across multiple species if working with non-human models

  • The antibody has been validated in human, mouse, and rat samples

Implementing these controls allows for confident interpretation of experimental results and helps distinguish specific signals from background or artifacts.