HIST1H3A (Ab-128) Antibody

What is the HIST1H3A (Ab-128) antibody and what epitope does it recognize?

The HIST1H3A (Ab-128) is a rabbit polyclonal antibody that specifically recognizes a peptide sequence around the Arginine 128 site of human Histone H3.1. This antibody targets one of the core histone proteins essential for chromatin structure and gene regulation . The antibody has been validated for multiple applications including ELISA, Western blotting, immunohistochemistry, and immunoprecipitation, making it versatile for various experimental approaches .

What are the validated applications for HIST1H3A (Ab-128) antibody?

The HIST1H3A (Ab-128) polyclonal antibody has been validated for the following applications:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB)

• Immunohistochemistry (IHC)

• Immunoprecipitation (IP)

For western blotting applications, this antibody provides high affinity binding with minimal background, allowing for clear detection of the target protein in human samples . When used for immunohistochemistry, this antibody can effectively detect histone H3 in paraffin-embedded tissue sections after appropriate antigen retrieval, similar to what has been demonstrated with related histone H3 antibodies .

What species reactivity has been confirmed for this antibody?

The HIST1H3A (Ab-128) antibody has been specifically validated for reactivity with human (Homo sapiens) samples . While this antibody specifically targets human histone H3.1, researchers should note that histone proteins are highly conserved across species, which may allow for cross-reactivity with other mammalian species, though additional validation would be required for such applications.

What is the recommended protocol for using HIST1H3A (Ab-128) antibody in Western blotting?

For optimal results in Western blotting applications using the HIST1H3A (Ab-128) antibody, follow this methodological approach:

• Prepare whole cell lysates or nuclear extracts containing histone proteins

• Separate proteins using SDS-PAGE (15-18% gels recommended for histone proteins)

• Transfer proteins to PVDF or nitrocellulose membrane

• Block membrane with 5% non-fat milk or BSA in TBST

• Incubate with HIST1H3A (Ab-128) antibody at an optimal dilution (typically 1:1000-1:2000)

• Incubate overnight at 4°C for best results

• Wash with TBST buffer (3-5 times)

• Apply appropriate HRP-conjugated secondary antibody

• Detect using enhanced chemiluminescence

This antibody has been successfully tested on human HeLa cell lysates and other human cell lines, similar to what has been reported with related histone H3 antibodies . The expected molecular weight for histone H3 is approximately 15-17 kDa.

What antigen retrieval method is recommended for immunohistochemistry with this antibody?

For optimal immunohistochemical detection using the HIST1H3A (Ab-128) antibody:

• Use heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

• Block tissue sections with 10% goat serum

• Incubate with primary antibody at 2 μg/ml concentration overnight at 4°C

• Apply peroxidase-conjugated secondary antibody for 30 minutes at 37°C

• Develop using a DAB chromogen system

This protocol is similar to the validated methodology for related histone H3 antibodies that have shown specific nuclear staining in human tissue sections, including esophageal squamous carcinoma and lung cancer tissues . The HIST1H3A antibody should produce distinct nuclear staining patterns consistent with the localization of histone proteins within the nucleus.

How should samples be prepared for immunoprecipitation with HIST1H3A (Ab-128) antibody?

For effective immunoprecipitation using HIST1H3A (Ab-128) antibody:

• Prepare nuclear extracts under non-denaturing conditions

• Pre-clear lysates with protein A/G beads

• Incubate cleared lysates with 2-5 μg of HIST1H3A antibody overnight at 4°C

• Add protein A/G beads and incubate for 1-2 hours

• Wash extensively with IP buffer containing low concentrations of detergent

• Elute immunoprecipitated complexes

• Analyze by Western blotting

This approach is particularly useful for studying histone H3 post-translational modifications or protein interactions, such as those involved in the formation of beta-catenin:tcf transactivating complexes . The antibody's specificity for the Arg-128 region makes it valuable for studying C-terminal interactions of histone H3.

How can HIST1H3A (Ab-128) antibody be used to study histone H3 mutations in cancer research?

For cancer research applications, the HIST1H3A (Ab-128) antibody can be utilized in multi-faceted approaches:

• Differential expression analysis: Compare histone H3.1 levels between normal and cancerous tissues using Western blotting or IHC

• Chromatin studies: Combine with ChIP assays to examine histone H3 distribution at specific genomic loci

• Mutation analysis: Use alongside mutant-specific antibodies to compare wild-type vs. mutant histone distribution

This approach is particularly relevant for pediatric high-grade gliomas and chondrosarcomas where histone H3 mutations (such as K27M and K36M) play crucial roles in tumorigenesis . The antibody can help distinguish between wild-type H3 and mutant forms when used in combination with mutation-specific antibodies.

Can this antibody detect post-translational modifications of histone H3?

The HIST1H3A (Ab-128) antibody recognizes the region around Arginine 128, which is distinct from the N-terminal tail where most post-translational modifications (PTMs) occur. Therefore:

• This antibody detects total histone H3.1 regardless of most N-terminal modifications

• It is ideal as a loading control when studying various histone H3 modifications

• For specific PTM detection, researchers should use modification-specific antibodies (e.g., anti-H3K27me3, anti-H3K9me3)

When studying specific modifications like H3K9 trimethylation mediated by SETDB1, which has implications in leukemia progression , this antibody can serve as a control to normalize for total H3 levels while using modification-specific antibodies to detect the methylation status.

How can HIST1H3A (Ab-128) be used to investigate SETDB1-mediated histone H3 lysine 9 methylation in leukemia?

To investigate SETDB1-mediated H3K9 methylation in leukemia research:

• Perform co-immunoprecipitation using HIST1H3A (Ab-128) antibody to pull down H3.1-containing complexes

• Analyze immunoprecipitated material for SETDB1 association and H3K9 methylation status

• Conduct ChIP-qPCR at HOXA9 and MEIS1 loci using this antibody in parallel with H3K9me3-specific antibodies

• Compare histone methylation patterns between samples with normal vs. overexpressed SETDB1

This methodological approach would help elucidate how SETDB1-mediated H3K9 methylation suppresses MLL-AF9 leukemia through regulation of HOXA9 and MEIS1 expression . The data would reveal correlations between H3.1 distribution, methylation status, and oncogene expression in leukemia models.

What are potential causes for high background when using HIST1H3A (Ab-128) antibody in immunohistochemistry?

When encountering high background in IHC applications, consider these methodological solutions:

• Optimize blocking conditions:

  • Increase blocking time or concentration (try 10-15% serum)

  • Consider alternative blocking agents (BSA, casein, or commercial blocking solutions)

• Adjust antibody concentration:

  • Conduct a titration series (1:500, 1:1000, 1:2000, etc.)

  • Reduce primary antibody concentration if background persists

• Modify washing steps:

  • Increase number and duration of washes

  • Add 0.1-0.3% Triton X-100 to wash buffers to reduce non-specific binding

• Optimize antigen retrieval:

  • Test different retrieval buffers (citrate pH 6.0 vs. EDTA pH 8.0)

  • Adjust retrieval time and temperature

Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has been shown to be effective for related histone H3 antibodies in human tissue sections , and similar conditions should work well for HIST1H3A (Ab-128) antibody.

How can I distinguish between HIST1H3A signal and other histone H3 variants?

Distinguishing between highly similar histone H3 variants requires careful experimental design:

• Compare immunoblotting patterns with variant-specific antibodies

• Perform knockdown validation:

  • Use siRNA targeting specific H3 variants

  • Confirm signal reduction by Western blot or IHC

• Mass spectrometry analysis:

  • Immunoprecipitate with HIST1H3A (Ab-128)

  • Analyze by mass spectrometry to identify captured variants

  • Quantify relative abundance of each variant

• ChIP-seq analysis:

  • Compare genomic distribution patterns with variant-specific antibodies

  • Look for distinct localization patterns that differentiate H3.1 from H3.3

The HIST1H3A (Ab-128) antibody is generated against a peptide sequence around Arg-128 of human histone H3.1 , but due to high sequence conservation among H3 variants, cross-reactivity may occur and should be carefully controlled for in experiments requiring variant specificity.

What are the expected staining patterns for HIST1H3A (Ab-128) antibody in normal versus cancer tissues?

Expected staining pattern comparison:

Interpretation guidelines:

• Nuclear localization is expected as histone H3 is a core component of nucleosomes

• Intensity variations may correlate with proliferation status and chromatin state

• Altered patterns in cancer may reflect changes in chromatin structure and histone modifications associated with malignancy

Based on studies with related histone H3 antibodies, specific nuclear staining has been observed in both esophageal squamous carcinoma and lung cancer tissues following appropriate antigen retrieval procedures .

How can HIST1H3A (Ab-128) antibody be incorporated into ChIP-seq experiments?

For optimal ChIP-seq experiments using HIST1H3A (Ab-128) antibody:

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

• Sonicate chromatin to fragments of 200-500 bp

• Immunoprecipitate using 2-5 μg of HIST1H3A (Ab-128) antibody per sample

• Include appropriate controls:

  • Input chromatin

  • IgG control

  • Positive control (antibody against abundant histone mark)

• Wash stringently to remove non-specific binding

• Reverse cross-links and purify DNA

• Prepare libraries for next-generation sequencing

This approach will allow mapping of histone H3.1 distribution across the genome, which can be particularly informative when comparing normal cells to cancer cells with H3 mutations . The resulting data can reveal changes in nucleosome positioning and chromatin accessibility that may contribute to altered gene expression patterns.

Can HIST1H3A (Ab-128) antibody be used effectively in multiplex immunofluorescence with other histone modification antibodies?

For multiplex immunofluorescence applications:

• Select compatible secondary antibodies with distinct fluorophores

• Optimize antibody dilutions for each primary antibody individually

• Consider sequential staining protocol:

  • Apply first primary antibody (e.g., HIST1H3A)

  • Detect with secondary antibody

  • Block remaining binding sites

  • Apply second primary antibody (e.g., H3K9me3)

  • Detect with different fluorophore-conjugated secondary

This approach is valuable for studying the relationship between histone H3 distribution and specific modifications, such as H3K9 methylation in the context of SETDB1 activity . Colocalization analysis can reveal regions where H3.1 is enriched with specific modifications versus regions where it lacks certain marks.

What flow cytometry protocol is recommended for intracellular staining with HIST1H3A (Ab-128) antibody?

For intracellular flow cytometry using HIST1H3A (Ab-128) antibody:

• Fix cells with 4% paraformaldehyde for 15 minutes

• Permeabilize with 0.1-0.3% Triton X-100 or commercial permeabilization buffer

• Block with 5% BSA or FBS

• Incubate with HIST1H3A (Ab-128) antibody (1:100-1:500 dilution)

• Wash thoroughly

• Incubate with fluorophore-conjugated secondary antibody

• Analyze by flow cytometry

This method allows quantification of histone H3 levels across cell populations and can be combined with cell cycle analysis or other markers to investigate relationships between histone H3 dynamics and cellular processes. Similar approaches have been validated for related histone H3 antibodies in flow cytometry applications .