HIST1H1D (Ab-140) Antibody

What is HIST1H1D and what biological role does it play?

HIST1H1D, also known as Histone H1.3 (Histone H1c or Histone H1s-2), is a member of the histone H1 family that plays a crucial role in chromatin organization and gene regulation. Histone H1 proteins bind to linker DNA between nucleosomes, forming the macromolecular structure known as the chromatin fiber. These proteins are necessary for the condensation of nucleosome chains into higher-order structured fibers. Beyond structural roles, HIST1H1D functions as a regulator of individual gene transcription through mechanisms including chromatin remodeling, nucleosome spacing, and DNA methylation. The protein has been implicated in various cellular processes related to epigenetic regulation, with aberrant expression or function linked to diseases such as cancer and neurological disorders .

What are the technical specifications of the HIST1H1D (Ab-140) Antibody?

The HIST1H1D (Ab-140) Antibody is a polyclonal antibody produced in rabbits that specifically targets the human Histone H1.3 protein. It is generated using a synthetic peptide sequence around the site of Lys (140) derived from human Histone H1.3 protein. The antibody is supplied in liquid form in a buffer containing 0.01 M PBS (pH 7.4), 0.03% Proclin-300, and 50% Glycerol. It has been purified using antigen affinity chromatography methods to ensure high specificity .

The antibody has been validated for several applications including ELISA, Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence/Immunocytochemistry (IF/ICC). Recommended dilutions vary by application: 1:100-1:1000 for WB, 1:20-1:200 for IHC, and 1:50-1:200 for IF/ICC. The antibody belongs to the IgG isotype and is unconjugated in its standard form .

How does the HIST1H1D (Ab-140) Antibody differ from other histone H1 antibodies?

The HIST1H1D (Ab-140) Antibody is distinguished by its specific targeting of the lysine 140 region of the human histone H1.3 protein. This epitope specificity allows researchers to examine this particular histone variant as opposed to pan-H1 antibodies that recognize multiple H1 subtypes. The antibody's specificity for HIST1H1D (H1.3) makes it valuable for research distinguishing between different histone H1 family members, which can have distinct functions despite their structural similarities .

While many commercial antibodies target conserved regions of histone proteins, the HIST1H1D (Ab-140) antibody's focus on the Lys-140 region allows for more precise studies of modifications and interactions specific to this site. This can be particularly important in epigenetic studies where the exact position of modifications can dramatically affect chromatin structure and gene expression patterns .

What are the validated applications for HIST1H1D (Ab-140) Antibody and their optimal protocols?

The HIST1H1D (Ab-140) Antibody has been validated for multiple experimental applications, each with specific optimized protocols:

Western Blotting (WB):

• Recommended dilution: 1:100-1:1000

• Positive detection in multiple cell lines including Jurkat, K562, and PC-3 whole cell lysates

• Secondary antibody: Goat polyclonal to rabbit IgG at 1:50000 dilution

• Expected molecular weight: ~23 kDa

• Sample preparation: Nuclear extracts prepared using standard nuclear extraction kits are preferred for histone proteins

Immunohistochemistry (IHC):

• Recommended dilution: 1:20-1:200

• Antigen retrieval methods should be optimized based on fixation method

• Detection systems compatible with rabbit IgG antibodies are suitable

• Controls should include tissues known to express HIST1H1D

Immunofluorescence/Immunocytochemistry (IF/ICC):

• Recommended dilution: 1:50-1:200

• Fixation: 4% paraformaldehyde recommended for nuclear proteins

• Nuclear counterstaining with DAPI is advised for co-localization studies

• Permeabilization is critical for nuclear protein detection

ELISA:

• Recommended dilution: 1:2000-1:10000

• Immobilized antigen approach preferred for histone proteins

• Secondary detection systems compatible with rabbit IgG antibodies

How should samples be prepared for optimal HIST1H1D detection in different applications?

Sample preparation varies significantly by application and requires careful consideration to maintain histone integrity while ensuring accessibility:

For Western blotting:
Nuclear extraction is critical for histone protein analysis. Use dedicated nuclear extraction kits containing protease inhibitor cocktails to prevent degradation. When working with histones, specialized extraction procedures using acidic conditions (e.g., 0.2N HCl or 0.4N H₂SO₄) may improve extraction efficiency. These samples should be neutralized before SDS-PAGE loading. For HIST1H1D specifically, nuclear extracts have been successfully used in published studies .

For Immunohistochemistry:
Tissues should be fixed in 10% neutral buffered formalin or 4% paraformaldehyde and embedded in paraffin. Antigen retrieval is crucial for histone detection, with heat-induced epitope retrieval in citrate buffer (pH 6.0) often providing good results. For HIST1H1D detection, section thickness of 4-6 μm is recommended with deparaffinization and rehydration following standard protocols .

For Immunofluorescence:
Cells should be fixed with 4% paraformaldehyde for 15-20 minutes at room temperature, followed by permeabilization with 0.1-0.5% Triton X-100 for nuclear protein access. Blocking with 1-5% BSA or normal serum is recommended before antibody incubation. Washing steps should be thorough to minimize background signal .

What are the most reliable controls to include when using HIST1H1D (Ab-140) Antibody?

Robust experimental design requires multiple controls to ensure reliable interpretation of results:

Positive Controls:

• Cell lines with confirmed HIST1H1D expression (e.g., Jurkat, K562, PC-3)

• Recombinant HIST1H1D protein for western blot standardization

• Normal tissues with known expression patterns of histone H1.3

Negative Controls:

• Primary antibody omission control to assess secondary antibody specificity

• Isotype control (rabbit IgG) at the same concentration to evaluate non-specific binding

• Peptide competition/blocking with immunizing peptide to confirm epitope specificity

• HIST1H1D-knockdown cell lines when available

Loading/Housekeeping Controls:

• For western blotting, standard nuclear protein markers such as Lamin B

• For IHC and IF, adjacent section staining with established nuclear markers

• When studying posttranslational modifications, total HIST1H1D detection alongside modified forms

What are common issues when working with HIST1H1D (Ab-140) Antibody and how can they be resolved?

Several technical challenges may arise when working with HIST1H1D antibodies:

High Background Signal:

• Issue: Non-specific binding in immunostaining applications

• Solutions: (1) Increase blocking time/concentration (5% BSA or normal serum for 1-2 hours), (2) Reduce primary antibody concentration, (3) Increase wash duration and frequency (4-5 washes of 5 minutes each), (4) Ensure secondary antibody compatibility and specificity, (5) For IHC, quench endogenous peroxidase activity with H₂O₂ before antibody incubation

Weak or No Signal:

• Issue: Insufficient antigen detection

• Solutions: (1) Optimize antigen retrieval methods (test different buffers and heat conditions), (2) Increase antibody concentration, (3) Extend incubation time (overnight at 4°C), (4) Ensure proper sample preservation and storage, (5) For histones specifically, verify extraction efficiency with histone-specific extraction protocols

Non-specific Bands in Western Blot:

• Issue: Detection of proteins other than HIST1H1D

• Solutions: (1) Increase blocking stringency, (2) Use gradient gels for better separation of histone proteins, (3) Verify sample preparation methods are appropriate for nuclear proteins, (4) Consider using more specific detection systems, (5) Validate with peptide competition assays

How do different sample preparation methods affect HIST1H1D detection and what modifications might be necessary?

Sample preparation significantly impacts histone detection:

Fixation Effects:
Cross-linking fixatives like formaldehyde can mask epitopes, particularly in histone proteins which are rich in lysine residues. If weak signal is observed with the HIST1H1D (Ab-140) Antibody in fixed samples, consider alternative fixation methods such as methanol/acetone or shorter formaldehyde fixation times. For the HIST1H1D (Ab-140) Antibody specifically, which targets a lysine-containing epitope, overfixation can be particularly problematic .

Extraction Methods:
Standard cell lysis buffers may not efficiently extract histones due to their tight association with DNA. Specialized histone extraction protocols using acidic conditions (0.2N HCl or 0.4N H₂SO₄) better solubilize histones for analysis. For Western blotting applications with HIST1H1D (Ab-140) Antibody, nuclear extraction followed by acid extraction may provide optimal results. The use of protease inhibitors and working at cold temperatures is essential to prevent degradation .

Protein Modification Considerations:
Since HIST1H1D undergoes numerous post-translational modifications, sample preparation methods should preserve these modifications if they are being studied. For example, phosphatase inhibitors should be included when studying phosphorylation states, and deacetylase inhibitors when studying acetylation. Since the HIST1H1D (Ab-140) Antibody targets a lysine residue (Lys-140), be aware that modifications at this site might affect antibody recognition .

What strategies can improve reproducibility when working with HIST1H1D (Ab-140) Antibody across different experimental batches?

Ensuring reproducibility requires systematic approaches:

Antibody Validation and Storage:

• Validate each new antibody lot against a reference standard

• Aliquot antibodies to avoid repeated freeze-thaw cycles

• Store at -20°C as recommended by manufacturers

• Document lot numbers and maintain consistency when possible within a study

Standardized Protocols:

• Develop detailed standard operating procedures (SOPs) for each application

• Include positive and negative controls in every experiment

• Maintain consistent sample preparation methods throughout a study

• When changing any protocol component, run parallel validations with established methods

Quantitative Approaches:

• Use internal loading controls appropriate for nuclear proteins

• Consider implementing chromatin immunoprecipitation (ChIP) normalization strategies

• For western blotting, include concentration standards on each blot

• Document imaging parameters and analysis settings for densitometry or fluorescence quantification

How can HIST1H1D (Ab-140) Antibody be used to study histone post-translational modifications and chromatin dynamics?

The HIST1H1D (Ab-140) Antibody can be leveraged for advanced studies of histone biology:

Chromatin Immunoprecipitation (ChIP):
While not explicitly listed in the provided validation data, many histone antibodies can be adapted for ChIP applications. For the HIST1H1D (Ab-140) Antibody, ChIP protocols would need optimization. Typically, this would involve crosslinking chromatin with formaldehyde, sonication to fragment DNA, immunoprecipitation with the antibody, washing, reverse crosslinking, and analysis of associated DNA sequences. This approach can reveal genomic regions where HIST1H1D is enriched, providing insights into its regulatory functions .

Co-Immunoprecipitation for Protein Complexes:
The antibody can be used to identify protein interaction partners of HIST1H1D through co-immunoprecipitation followed by mass spectrometry or western blotting. This approach has revealed interactions between histone H1 variants and chromatin remodeling factors. Recent research has identified WHSC1 as a HIST1H1D-interacting protein that monomethylates H1 at K85, demonstrating how such approaches can identify novel modifications and interactions .

Multiplex Immunofluorescence:
Combining HIST1H1D (Ab-140) Antibody with antibodies against other chromatin-associated proteins or specific histone modifications can reveal spatial relationships within the nucleus. This approach can demonstrate whether HIST1H1D co-localizes with heterochromatin or euchromatin markers, or with specific transcriptional regulators .

What is the current understanding of the relationship between HIST1H1D modifications and gene expression regulation?

Histone H1 variants, including HIST1H1D, play sophisticated roles in gene regulation:

Methylation Effects:
Research has shown that WHSC1 monomethylates histone H1 at K85, and this modification may influence stem cell-like features in cancer cells. This demonstrates that specific modifications to H1 histones can have significant biological consequences. While less studied than core histone modifications, H1 modifications represent an emerging area of epigenetic regulation .

Role in Chromatin Compaction:
HIST1H1D contributes to higher-order chromatin structure through its binding to linker DNA. The modification state of HIST1H1D can influence its binding affinity and thus affect chromatin accessibility. Regions with reduced HIST1H1D binding often correlate with active gene expression, while regions enriched in HIST1H1D typically show repressed transcription .

Interaction with Other Epigenetic Mechanisms:
HIST1H1D functions within a complex network of epigenetic regulators. Research suggests that H1 variants, including HIST1H1D, can influence DNA methylation patterns and the distribution of other histone modifications such as H3K27me3 and H3K4me3. These interactions create a sophisticated regulatory system controlling gene expression in different cellular contexts .

How does HIST1H1D contribute to disease mechanisms and what are the implications for therapeutic development?

Understanding HIST1H1D's role in disease has significant implications:

Cancer Biology:
Abnormal expression or modification of HIST1H1D has been observed in various cancers. Research has shown that WHSC1-mediated H1 monomethylation may enhance stemness features in squamous cell carcinoma of the head and neck (SCCHN), suggesting a role in cancer progression. This finding indicates that targeting specific modifications of histone H1 variants could represent a novel therapeutic approach .

Immune System Regulation:
Research has identified histone-reactive B cells that recognize histone proteins, including H1 variants. These autoreactive B cells are typically maintained in an anergic state in healthy individuals but may contribute to autoimmune conditions when tolerance mechanisms fail. Interestingly, some histone-reactive antibodies have been shown to neutralize HIV-1, suggesting complex relationships between autoreactivity and anti-pathogen immunity .

Epigenetic Dysregulation:
As a regulator of chromatin structure, altered HIST1H1D function can contribute to broad epigenetic dysregulation. Studies of histone acylations (including acetylation, crotonylation, and butyrylation) have expanded our understanding of the epigenetic code, with implications for numerous diseases characterized by epigenetic abnormalities. The HIST1H1D (Ab-140) Antibody can be valuable in investigating these mechanisms .

What quantitative methods are recommended for analyzing HIST1H1D expression or localization data?

Rigorous quantification enhances the value of HIST1H1D studies:

Western Blot Densitometry:
For quantifying HIST1H1D protein levels, densitometric analysis of western blots provides relative quantification. Signal intensity should be normalized to appropriate nuclear loading controls such as Lamin B. Software packages like ImageJ/Fiji, Image Lab, or specialized commercial solutions can provide consistent analysis. To ensure accuracy, maintain the dynamic range of detection and include concentration standards on each blot .

Immunofluorescence Quantification:
For subcellular localization studies, quantitative image analysis should:

• Measure nuclear intensity of HIST1H1D staining across multiple cells (n>30)

• Normalize to nuclear area or DNA content (DAPI signal)

• Assess co-localization with other markers using Pearson's correlation coefficient or Manders' overlap coefficient

• Use consistent imaging parameters and analysis thresholds across all samples

ChIP-Seq Data Analysis:
For genome-wide localization:

• Apply standard peak-calling algorithms (MACS2, HOMER)

• Normalize to input controls and total read depth

• Compare binding patterns to known genomic features (promoters, enhancers)

• Integrate with gene expression data to correlate binding with transcriptional outcomes

How can researchers distinguish between HIST1H1D and other histone H1 variants in their experiments?

Distinguishing between highly similar histone variants requires specific approaches:

Antibody Specificity Verification:
The HIST1H1D (Ab-140) Antibody targets a specific region around Lys-140 of human Histone H1.3. To confirm specificity:

• Perform peptide competition assays with the immunizing peptide

• Test reactivity against recombinant H1 variants

• Verify absence of signal in HIST1H1D knockout or knockdown samples

• Consider mass spectrometry validation in immunoprecipitation experiments

Complementary Molecular Approaches:

• RT-qPCR can distinguish variant-specific mRNA expression using primer sets targeting unique regions

• Targeted mass spectrometry can identify peptides unique to HIST1H1D

• Variant-specific knockdown/knockout followed by rescue experiments with other variants can reveal functional specificity

Bioinformatic Analysis:
For ChIP-seq or similar data:

• Compare binding profiles to published datasets of other H1 variants

• Identify HIST1H1D-specific binding regions for further validation

• Correlate with known functional differences between H1 variants based on literature

What are the key considerations when interpreting results from different applications using HIST1H1D (Ab-140) Antibody?

Critical interpretation requires understanding the limitations and context of each technique:

Western Blot Interpretation:

• Molecular weight confirmation: HIST1H1D should appear at approximately 23 kDa

• Multiple bands may indicate post-translational modifications, degradation products, or non-specific binding

• Nuclear extraction quality significantly impacts results; poor extraction may yield false negatives

• When studying modifications, consider using modification-specific antibodies alongside the HIST1H1D (Ab-140) Antibody

Immunostaining Pattern Analysis:

• HIST1H1D typically shows nuclear localization with potential heterogeneity in staining intensity

• Distribution patterns may vary with cell cycle phase or cell type

• Co-localization with heterochromatin markers (e.g., H3K9me3) may indicate functional state

• Careful interpretation is needed when comparing normal vs. diseased tissues, as both expression levels and localization patterns may change

Cross-Reactivity Considerations:
Recent research has highlighted cross-reactivity issues with some pan-K-acyl antibodies in epigenetics research. When studying histone modifications, it's crucial to validate antibody specificity through multiple approaches. The HIST1H1D (Ab-140) Antibody should be validated for specificity to ensure it's not cross-reacting with other H1 variants or detecting non-specific proteins, especially in complex samples .