HIST1H2BC (Ab-116) Antibody

What is HIST1H2BC and what role does it play in cellular biology?

HIST1H2BC (Histone H2B type 1-C) is a histone protein that plays a crucial role in chromatin structure and organization. It is involved in the regulation of gene expression, DNA repair, transcription, and cell differentiation. As part of the nucleosome core, HIST1H2BC helps package DNA in eukaryotic cells, making it essential for maintaining genome integrity and controlling gene activity . The protein undergoes various post-translational modifications that contribute to the "histone code," which regulates chromatin accessibility and transcriptional activity. Aberrant expression or function of histone proteins, including HIST1H2BC, can lead to various diseases, including cancer and developmental disorders .

What are the key specifications of HIST1H2BC (Ab-116) Antibody products?

HIST1H2BC (Ab-116) Antibody products such as PACO60509 and PACO59653 share the following specifications:

These antibodies are designed for research purposes and have been validated for multiple experimental applications .

What is the molecular weight of HIST1H2BC protein and how can I confirm antibody specificity?

The calculated molecular weight of HIST1H2BC protein is approximately 13.9 kDa . To confirm antibody specificity, researchers should perform validation experiments including:

• Western blotting with positive controls (e.g., Hela, K562, HL60, HepG2, or 293 whole cell lysates have shown positive results)

• Including negative controls (cells/tissues not expressing the target)

• Peptide competition assays to confirm binding specificity

• Cross-reactivity testing with related histone variants

The UniProt accession number for HIST1H2BC is P62807, with secondary accession numbers P02278, Q3B872, Q4VB69, Q93078, and Q93080 . Comparing your experimental results with known expression patterns can provide additional validation of antibody specificity.

How should I design experiments using HIST1H2BC antibodies to avoid batch effects?

When designing experiments with HIST1H2BC antibodies, careful planning is essential to avoid batch effects that can confound your results:

• Process all samples at the same time when feasible, or minimize confounding through blocking and randomization

• Include controls for each experimental batch

• Document all potential batch variables (processing dates, reagent lots, operators)

• If complete synchronous processing is impossible, ensure treatment and control samples are distributed across batches rather than segregated

In statistical modeling, batch effects can be included as covariates in the model. For exploratory analysis, attempt to "eliminate" or "adjust for" unwanted variation in advance by subtracting the estimated effect from each variable . Remember that even partial confounding between batch and signal of interest can lead to bias in your results.

What are the optimal storage conditions for HIST1H2BC antibodies to maintain activity?

To maintain optimal activity of HIST1H2BC antibodies:

• Store antibody aliquots at -20°C for long-term storage

• Avoid repeated freeze/thaw cycles by preparing working aliquots

• For short-term use (up to one month), storage at 4°C is acceptable

• Ensure the antibody remains in its recommended buffer (typically 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4)

• Keep antibody vials tightly sealed and protected from light

• Monitor expiration dates and periodically validate activity with positive controls

Following these practices will help maintain antibody specificity and sensitivity throughout your research project.

How can I optimize Western blot protocols specifically for HIST1H2BC detection?

Optimizing Western blot protocols for HIST1H2BC detection requires several specific considerations:

• Sample preparation:

  • Use specialized histone extraction protocols that include acid extraction to efficiently isolate histones

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

  • Use fresh samples when possible

• Gel electrophoresis:

  • Use high percentage (15-18%) gels for better resolution of small proteins (HIST1H2BC is 13.9 kDa)

  • Consider using specialized gel systems designed for low molecular weight proteins

  • Load appropriate positive controls (e.g., Hela, K562, HL60, HepG2, or 293 whole cell lysates)

• Transfer and detection:

  • Use PVDF membranes with 0.2 μm pore size rather than 0.45 μm for better retention of small proteins

  • Optimize antibody dilution (start with 1:100-1:1000 as recommended)

  • Extend incubation times (overnight at 4°C) for better signal

  • Use enhanced chemiluminescence detection with sensitive substrates

• Troubleshooting:

  • If you detect multiple bands, consider cross-reactivity with other histone H2B variants

  • Verify signal with peptide competition assay

  • Adjust blocking conditions if high background is observed

What techniques can be used to study HIST1H2BC post-translational modifications?

Studying post-translational modifications (PTMs) of HIST1H2BC requires specialized techniques:

• Mass spectrometry-based approaches:

  • Time-lapse enzymatic deacetylation (HDAC1) combined with MS analysis can help study acetylation on histones

  • This approach allows for quantify-first strategy that isolates ion populations of interest

  • When optimized, this method can double the portion of annotated precursors of interest (from 10.5% to 21.6%)

• Chromatin immunoprecipitation (ChIP):

  • Use HIST1H2BC antibodies in combination with antibodies against specific modifications

  • ChIP-seq can map genome-wide distribution of modified HIST1H2BC

• Specialized Western blotting:

  • Use modification-specific antibodies alongside general HIST1H2BC antibodies

  • Compare patterns before and after treatment with modifying or demodifying enzymes

• Immunofluorescence microscopy:

  • Co-staining with modification-specific antibodies can reveal spatial distribution of modified HIST1H2BC

These approaches allow researchers to investigate the "histone code" involving HIST1H2BC and its role in gene regulation and chromatin organization.

What are the challenges in MS-based analysis of HIST1H2BC and how can they be addressed?

MS-based analysis of HIST1H2BC faces several challenges that require specific approaches:

• Combinatorial complexity:

  • Histone posttranslational modifications (hPTMs) create an alphabet with more letters than amino acids, resulting in a "histone code" with extremely high combinatorial complexity

  • A significant portion of acquired MSMS spectra remains unannotated due to this complexity

• False discovery rate control:

  • Lack of validated FDR control makes it difficult to assess whether newly assigned ions are informative

  • Time-lapse enzymatic deacetylation (HDAC1) of commercial histone extract provides a quantify-first strategy to isolate ion populations of interest

• Sample preparation issues:

  • Histone extraction methods can affect PTM preservation

  • Use specialized extraction protocols designed for histones

• Data acquisition and analysis:

  • Adapt search parameters to study potential issues in sample preparation, data acquisition, and data analysis

  • This stepwise approach can significantly increase the portion of annotated precursors (from 10.5% to 21.6%)

• Solutions:

  • Use the time-lapse enzymatic approach to make up for the lack of validated FDR control

  • Implement workflow adaptations that reduce the portion of the dark histone ion space

  • Apply the strategy with various enzymes targeting modifications of interest

How does HIST1H2BC (Ab-116) antibody performance compare across different experimental techniques?

The performance of HIST1H2BC (Ab-116) antibody varies across experimental techniques:

• Western Blotting (WB):

  • Recommended dilution: 1:100-1:1000

  • Shows positive results with multiple cell lines (HeLa, K562, HL60, HepG2, 293)

  • Good sensitivity for detecting the 13.9 kDa protein

  • Specificity confirmed through various control experiments

• Immunohistochemistry (IHC):

  • Recommended dilution: 1:10-1:100

  • Requires careful optimization of antigen retrieval methods

  • May require signal amplification systems for low-abundance detection

  • Best results with formalin-fixed, paraffin-embedded tissues

• Immunofluorescence (IF):

  • Recommended dilution: 1:1-1:10 (note the higher concentration needed)

  • Provides spatial information about HIST1H2BC distribution

  • Can be combined with other markers for co-localization studies

  • May require confocal microscopy for detailed nuclear localization

• ELISA:

  • Recommended dilution: 1:2000-1:10000

  • Offers quantitative measurement capability

  • Higher sensitivity compared to other techniques

  • Useful for high-throughput screening

Researchers should validate the antibody in their specific experimental context and may need to adjust dilutions beyond the recommended ranges depending on sample type and detection system.

What control samples and validation techniques are recommended when using HIST1H2BC antibodies?

For rigorous research using HIST1H2BC antibodies, the following controls and validation techniques are recommended:

• Positive controls:

  • Cell lines with known HIST1H2BC expression (e.g., HeLa, K562, HL60, HepG2, 293)

  • Rat spleen tissue (for PACO59653 which reacts with rat)

  • Recombinant HIST1H2BC protein as a standard

• Negative controls:

  • Primary antibody omission

  • Isotype control (rabbit IgG)

  • Peptide competition/neutralization to confirm specificity

  • Tissues or cells with HIST1H2BC knockdown/knockout (if available)

• Validation techniques:

  • Molecular weight confirmation (expected ~13.9 kDa)

  • Cross-validation with multiple antibodies targeting different epitopes

  • Orthogonal techniques (e.g., mRNA expression correlation)

  • Reproducibility testing across different lots

• Experimental design controls:

  • Include biological replicates

  • Account for batch effects through proper experimental design

  • Include technical replicates for critical experiments

Employing these controls and validation techniques ensures research reliability and facilitates troubleshooting when unexpected results occur.

How can I interpret contradictory results when analyzing HIST1H2BC expression or modifications?

Contradictory results in HIST1H2BC research may arise from several factors:

• Technical considerations:

  • Antibody cross-reactivity with other histone H2B variants

  • Different extraction methods affecting histone recovery

  • Batch effects in sample processing

  • Different detection sensitivities across platforms

• Biological variables:

  • Cell cycle-dependent expression and modifications

  • Tissue-specific regulation

  • Response to cellular stress or experimental conditions

  • Impact of other epigenetic modifications

• Analytical approach:

  • Create a structured analysis flow to identify sources of variation

  • Document all experimental conditions meticulously

  • Use statistical approaches that account for batch effects

  • Consider that even partial confounding between batch and signal of interest can lead to bias

• Resolution strategies:

  • Perform time-course experiments to capture dynamic changes

  • Use multiple antibodies and detection methods

  • Incorporate controls for cell cycle stage

  • Implement single-cell approaches to detect heterogeneity

  • Use p-value histograms to evaluate batch effect impact on statistical analysis

What are the current research frontiers involving HIST1H2BC and how can the antibody contribute to these areas?

Current research frontiers involving HIST1H2BC include:

• Epigenetic regulation in disease:

  • HIST1H2BC modifications contribute to the "histone code" that regulates gene expression

  • Using the antibody to map HIST1H2BC distribution and modifications in disease models can reveal epigenetic dysregulation mechanisms

  • Applications in cancer, developmental disorders, and neurological conditions research

• Chromatin dynamics and 3D genome organization:

  • HIST1H2BC plays a role in higher-order chromatin structure

  • Antibodies enable chromatin immunoprecipitation studies to investigate DNA-protein interactions

  • Contributes to understanding how genome organization affects cellular function

• Histone code deciphering:

  • Advanced mass spectrometry approaches are being developed to better analyze histone PTMs

  • Time-lapse enzymatic deacetylation methods help isolate ion populations of interest

  • These approaches significantly increase the annotation of histone modifications previously in the "dark histone ion space"

• Single-cell epigenomics:

  • HIST1H2BC antibodies can be adapted for single-cell approaches to study epigenetic heterogeneity

  • Integration with other single-cell technologies provides comprehensive views of cellular states

• Therapeutic targeting:

  • Understanding HIST1H2BC modifications helps identify potential targets for epigenetic therapies

  • Antibodies provide tools to evaluate treatment efficacy in modulating histone modifications

The HIST1H2BC antibody serves as a crucial tool in these research areas, enabling detection, quantification, and functional characterization of this important histone variant and its modifications.