HIST1H2AC Antibody

What is HIST1H2AC and why is it significant in research?

HIST1H2AC (histone cluster 1, H2ac) is a specific replication-dependent histone H2A isoform encoded by the HIST1H2AC gene. Although histones are generally considered functionally redundant, HIST1H2AC has emerged as functionally distinct with significant implications in cellular processes. It is a 130 amino acid protein with a calculated molecular weight of 14 kDa, though it typically appears as a 17 kDa band in experimental settings .

The significance of HIST1H2AC in research stems from evidence showing its altered expression is associated with cancer development. Reduced expression of the HIST1H2AC locus leads to increased rates of cell proliferation and tumorigenicity, suggesting it may function as a tumor suppressor . Mass spectrometry analyses have identified HIST1H2AC as the second most abundant replication-dependent H2A isoform in HeLa cells, encoded by a single gene, unlike other abundant variants encoded by multiple genes . This unique attribute makes HIST1H2AC an important target for epigenetic and cancer research.

What applications are HIST1H2AC antibodies validated for?

HIST1H2AC antibodies have been validated for multiple experimental applications, with reactivity in human, mouse, and rat samples. Based on extensive validation data, these antibodies can be reliably used in:

When performing immunohistochemistry with HIST1H2AC antibodies, antigen retrieval is critical for optimal results. The recommended protocol suggests using TE buffer at pH 9.0, though citrate buffer at pH 6.0 may serve as an alternative . For Western blotting applications, HIST1H2AC antibodies have successfully detected the protein in mouse and rat liver tissues, with observed molecular weight at approximately 17 kDa .

How should HIST1H2AC antibodies be stored and handled?

Proper storage and handling of HIST1H2AC antibodies are essential for maintaining their reactivity and specificity. According to manufacturer guidelines, HIST1H2AC antibodies should be:

• Stored at -20°C where they remain stable for one year after shipment .

• Kept in storage buffer consisting of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Aliquoting is generally unnecessary for -20°C storage, reducing the risk of contamination from repeated freeze-thaw cycles .

• For antibody preparations containing BSA (such as those in 20μl sizes that contain 0.1% BSA), care should be taken to prevent bacterial contamination .

When working with azide and BSA-free formulations, additional precautions may be necessary. These preparations are typically shipped with polar packs and should be stored immediately at the recommended temperature upon receipt . To maintain antibody integrity, avoid repeated freeze-thaw cycles as these can lead to protein denaturation and loss of activity.

How can I validate the specificity of a HIST1H2AC antibody for my research?

Validating antibody specificity is critical for reliable research outcomes. For HIST1H2AC antibodies, a comprehensive validation approach should include:

Genetic validation: Implement siRNA knockdown targeting the HIST1H2AC gene specifically. Researchers have successfully used siRNA targeting the H2A 1C gene to confirm antibody specificity by observing decreased signal corresponding to reduced gene expression . When designing validation experiments, it's important to note that multiple H2A variants exist, so specificity of knockdown should be confirmed using real-time PCR assays targeting individual H2A isoform transcripts .

Immunogen analysis: Examine the immunogen sequence used to generate the antibody. For example, the 4F10 monoclonal antibody was generated against a partial recombinant HIST1H2AC protein (amino acids 25-96) with a GST tag . Understanding this sequence helps predict potential cross-reactivity with other histone variants.

Multiple technique verification: Confirm consistent results across different techniques. If an antibody shows expected results in both Western blot and immunohistochemistry applications with appropriate controls, this strengthens confidence in its specificity .

Known molecular weight verification: HIST1H2AC has a calculated molecular weight of 14 kDa but is typically observed at 17 kDa in experimental settings . This discrepancy is common for histones due to their post-translational modifications and should be considered when evaluating Western blot results.

What is the functional significance of HIST1H2AC in tumorigenesis?

The relationship between HIST1H2AC and tumorigenesis represents an emerging area of research with significant implications for cancer biology. Multiple lines of evidence suggest that HIST1H2AC plays a functional role in regulating cell proliferation and cancer development:

HIST1H2AC depletion studies have revealed that knockdown of H2A 1C using siRNA induces the formation of large colonies in soft agar assays, indicating increased tumorigenicity . This effect appears to be specific to H2A 1C, as knockdown of canonical H2A had a slightly negative impact on colony formation . These findings suggest that H2A 1C may function as a tumor suppressor.

Analysis of histone profiles across different cancer types has identified consistent alterations in HIST1H2AC abundance. In chronic lymphocytic leukemia (CLL), a decrease in the abundance of H2A 1C was observed in a high percentage of patient samples compared to healthy B cells . Similar changes were found in bladder cancer cell lines, with progressive decreases in HIST1H2AC abundance correlating with increased proliferation capacity and invasiveness .

The specificity of these effects to particular H2A isoforms challenges the conventional view that replication-dependent histones are functionally interchangeable. Instead, it suggests that HIST1H2AC may have unique regulatory functions in controlling cell proliferation pathways. The molecular mechanisms underlying these functions remain to be fully elucidated but may involve specific interactions with chromatin regulators or distinct effects on nucleosome dynamics.

How does HIST1H2AC regulation differ from other histone H2A variants?

HIST1H2AC exhibits unique regulatory mechanisms that distinguish it from other histone H2A variants:

Translational regulation: The 5′ UTR of HIST1H2AC mRNA contains distinctive elements that impart translational repression . This regulation appears to be specific to HIST1H2AC, as it is not observed in other H2A isoforms. Researchers have mapped this repression to a specific duplicated sequence element found only in the HIST1H2AC 5′ UTR .

Gene-specific expression control: Unlike other abundant H2A variants that are encoded by multiple genes, HIST1H2AC is encoded by a single gene . This unique arrangement potentially allows for more precise regulation of its expression levels. Experimental evidence using luciferase assays with 5′ UTRs of different histone H2A isoforms has demonstrated that the HIST1H2AC UTR specifically mediates translational control .

Differential abundance patterns in disease states: While multiple H2A variants show altered expression in cancer, HIST1H2AC shows particularly consistent patterns of decreased abundance across unrelated cancer types including CLL and bladder cancer . This suggests that HIST1H2AC may be subject to specific regulatory mechanisms that are commonly dysregulated during tumorigenesis.

RNA-binding protein interactions: Research has implicated RNA-binding proteins such as HuR in the regulation of HIST1H2AC expression. Experiments using HuR knockdown followed by luciferase assays with the HIST1H2AC 5′ UTR have helped elucidate this regulatory mechanism .

What are the optimal protocols for HIST1H2AC detection in different applications?

Western Blot Protocol:
When performing Western blot analysis with HIST1H2AC antibodies, researchers should consider the following optimization steps:

• Sample preparation: Use freshly prepared tissue lysates when possible. HIST1H2AC has been successfully detected in mouse and rat liver tissues .

• Loading control selection: When studying histones, traditional housekeeping proteins may not be ideal loading controls. Consider using total H2AX as a denominator for more reliable quantitation, especially when examining specific modifications .

• Dilution optimization: Start with the recommended dilution range (1:200-1:1000) and optimize based on signal-to-noise ratio .

• Expected molecular weight: Look for bands at approximately 17 kDa, despite the calculated molecular weight of 14 kDa .

Immunohistochemistry Protocol:
For IHC applications with HIST1H2AC antibodies:

• Antigen retrieval: Use TE buffer at pH 9.0 as the primary recommendation, with citrate buffer at pH 6.0 as an alternative .

• Antibody dilution: Begin with the recommended range of 1:50-1:500 and optimize for your specific tissue type .

• Positive control tissue selection: Human skin cancer tissue has been validated as a positive control for IHC with HIST1H2AC antibodies .

• Detection system selection: Choose a detection system compatible with rabbit IgG for polyclonal antibodies or mouse IgG for monoclonal antibodies, depending on the primary antibody used .

ELISA Protocol:
For quantitative analysis using ELISA:

• Optimal antibody concentration should be experimentally determined for each specific application .

• When using the 4F10 monoclonal antibody as a capture antibody, the detection limit for recombinant GST-tagged HIST1H2AC has been established at 0.1 ng/ml .

How can I troubleshoot issues with HIST1H2AC antibody experiments?

Common issues with HIST1H2AC antibody experiments and their solutions include:

Weak or no signal in Western blot:

• Increase antibody concentration within the recommended range (1:200-1:1000) .

• Extend primary antibody incubation time or temperature.

• Enhance antigen retrieval methods.

• Check sample preparation – HIST1H2AC is a nuclear protein requiring effective nuclear extraction.

• Verify protein transfer efficiency, especially for low molecular weight proteins like histones.

High background in immunohistochemistry:

• Optimize antibody dilution; start at the higher end of the recommended range (closer to 1:500) .

• Increase blocking time or concentration.

• Ensure thorough washing between steps.

• Reduce secondary antibody concentration.

• Try alternative antigen retrieval methods if the recommended TE buffer at pH 9.0 is causing excessive background .

Cross-reactivity concerns:

• Perform validation using siRNA knockdown of HIST1H2AC to confirm specificity .

• Consider using monoclonal antibodies like 4F10 that target specific epitopes when cross-reactivity is a concern .

• Include appropriate negative controls, such as isotype controls or samples known to be negative for HIST1H2AC.

Inconsistent results between experiments:

• Standardize tissue processing and fixation methods.

• Use consistent lot numbers of antibodies when possible.

• Include positive controls in each experiment.

• Ensure antibody storage conditions are maintained properly at -20°C .

How can HIST1H2AC antibodies be used to study cancer mechanisms?

HIST1H2AC antibodies offer valuable tools for investigating cancer mechanisms through several experimental approaches:

Differential expression analysis: HIST1H2AC abundance is altered in multiple cancer types. Using Western blot or immunohistochemistry with HIST1H2AC antibodies, researchers can quantify these changes across different cancer types and stages. Liquid chromatography-mass spectrometry (LC/MS) profiling of histones has revealed decreased abundance of H2A 1C in chronic lymphocytic leukemia (CLL) samples compared to healthy B cells, and similar changes were observed in bladder cancer cells with increasing invasiveness .

Functional studies: To investigate the role of HIST1H2AC in cancer development, researchers can combine HIST1H2AC knockdown using siRNA with functional assays. For example, after HIST1H2AC depletion, soft agar colony formation assays have revealed increased tumorigenicity . HIST1H2AC antibodies can be used to confirm successful knockdown at the protein level.

Chromatin immunoprecipitation (ChIP) assays: HIST1H2AC antibodies can be used in ChIP experiments to identify genomic regions where this specific histone variant is enriched, potentially revealing its role in regulating cancer-related genes.

Correlation with clinical outcomes: By analyzing HIST1H2AC levels in patient samples using immunohistochemistry, researchers can investigate correlations between HIST1H2AC abundance and clinical parameters such as disease progression, treatment response, and patient survival.

What are the current findings on HIST1H2AC expression in different cancer types?

Research has revealed distinct patterns of HIST1H2AC expression across different cancer types:

Chronic Lymphocytic Leukemia (CLL):
LC/MS profiling of histones from CLL patients showed a significant decrease in the abundance of H2A 1C and H2A 1B/E compared to CD19+ B cells from healthy individuals . This alteration was observed in a high percentage of CLL samples, suggesting it may be a common feature of this malignancy.

Bladder Cancer:
Analysis of histone H2A profiles in bladder cancer revealed a progressive decrease in HIST1H2AC abundance that correlated with:

• Initial acquisition of proliferative capacity in immortalized bladder epithelial cells

• Further decrease in non-invasive bladder cancer cells (RT4)

• Most pronounced decrease in invasive bladder cancer cells (T24)

This pattern suggests that reduction in HIST1H2AC abundance may be associated with bladder cancer progression and acquisition of invasive properties.

Experimental Validation:
The functional significance of decreased HIST1H2AC expression in cancer was experimentally validated using siRNA knockdown approaches. When HIST1H2AC expression was reduced in 293TN cells:

• Significant increase in colony formation was observed in soft agar assays

• The effect was specific to HIST1H2AC, as knockdown of canonical H2A had a slightly negative impact on colony formation

• Quantification using a fluorescent dye-based assay confirmed increased cell proliferation in soft agar following HIST1H2AC knockdown

These findings collectively suggest that HIST1H2AC may function as a tumor suppressor, with its decreased expression promoting cell proliferation and tumorigenicity across multiple cancer types.

What emerging techniques are enhancing HIST1H2AC research?

Emerging techniques are expanding the potential applications of HIST1H2AC antibodies in research:

Combined immunoassays: Integrating HIST1H2AC detection with other histone variants or modifications can provide more comprehensive insights. For example, the approach used for γH2AX and Total H2AX immunoassays, where total H2AX serves as a denominator for phosphorylated H2AX reporting, could be adapted for HIST1H2AC studies . This approach provides more reliable quantitation and a more relevant pharmacodynamic readout for monitoring treatment-induced changes.

CRISPR activation screening: Advanced genetic approaches such as CRISPR activation screening have been employed to identify downstream genes regulated by signaling pathways that may interact with HIST1H2AC. One publication cited in the HIST1H2AC antibody documentation used this technique in the context of NRAS signaling , suggesting similar approaches could illuminate HIST1H2AC regulatory networks.

Luciferase reporter assays: Specialized luciferase assays have been developed to study the unique regulatory elements in the 5′ UTR of HIST1H2AC. These assays involve cloning oligonucleotides corresponding to the 5′ UTR upstream of a luciferase reporter, enabling quantitative analysis of regulatory mechanisms specific to HIST1H2AC . This technique has proven valuable for understanding the translational repression uniquely associated with HIST1H2AC.

Mass spectrometry-based approaches: Advanced LC/MS profiling techniques have enabled precise quantification of histone variant abundance, revealing that HIST1H2AC is the second most abundant replication-dependent H2A isoform in HeLa cells . These techniques offer advantages in distinguishing between highly similar histone variants where antibody cross-reactivity might be a concern.

How can researchers integrate HIST1H2AC studies with broader epigenetic research?

Integrating HIST1H2AC research into broader epigenetic studies offers several promising avenues:

Histone code analysis: HIST1H2AC can be studied in the context of the histone code hypothesis by investigating how this specific variant interacts with various histone modifications and chromatin-associated proteins. Combining HIST1H2AC antibodies with antibodies against specific histone marks in co-immunoprecipitation or sequential ChIP experiments could reveal unique interactions.

Chromatin architecture studies: The functional consequences of altered HIST1H2AC expression on higher-order chromatin structure represent an important area for investigation. Techniques such as Hi-C or ATAC-seq combined with HIST1H2AC manipulation could reveal how this variant influences genome organization and accessibility.

Epigenetic therapy response: Given the altered expression of HIST1H2AC in cancer, investigating how epigenetic therapies affect HIST1H2AC levels and distribution could provide insights into mechanism of action and potentially identify biomarkers of treatment response.

Developmental epigenetics: Studying HIST1H2AC dynamics during development and differentiation could illuminate its role in establishing and maintaining cell type-specific epigenetic states. HIST1H2AC antibodies could be employed in ChIP-seq experiments across developmental time points or differentiation stages.