HIST1H2BC (Ab-116) Antibody

What is HIST1H2BC and why is it important for research?

HIST1H2BC (Histone H2B type 1-C) is a crucial histone variant involved in the regulation of chromatin structure and gene expression. Its study is significant because it plays fundamental roles in multiple cellular processes including DNA repair, transcription, and cell differentiation. The importance of HIST1H2BC extends to disease research, as aberrant histone modifications and expression have been implicated in various pathological conditions including cancer, developmental disorders, and neurological conditions. The HIST1H2BC (Ab-116) Antibody enables researchers to detect and analyze this protein across diverse experimental settings .

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

The HIST1H2BC (Ab-116) Antibody (PACO59653) is a rabbit-derived polyclonal antibody that specifically recognizes the peptide sequence around site of Lys (116) derived from Human Histone H2B type 1-C/E/F/G/I. It is supplied as a 50μl liquid preparation in a storage buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4. The antibody has been purified using antigen affinity methods, is non-conjugated, and belongs to the IgG isotype. It demonstrates reactivity with both human and rat specimens, making it suitable for cross-species comparative studies .

What applications has the HIST1H2BC (Ab-116) Antibody been validated for?

The antibody has been validated for multiple experimental applications including:

Positive Western blot detection has been confirmed in various whole cell lysates including HeLa, K562, HL60, HepG2, and 293, as well as in rat spleen tissue, demonstrating the antibody's versatility across different sample types .

How should I optimize Western blot analysis using HIST1H2BC (Ab-116) Antibody?

For optimal Western blot results with HIST1H2BC (Ab-116) Antibody, follow these methodological recommendations:

• Cell Lysis: Use a standard lysis buffer (e.g., 50mM Tris-HCl [pH 7.4], 150mM NaCl, 0.5% NP-40) as used in comparable histone protein analyses.

• Protein Separation: Resolve cell lysates on 4-15% gradient SDS-PAGE gels, which provide optimal separation for the ~14 kDa histone proteins.

• Transfer: Use nitrocellulose membranes for consistent results.

• Antibody Dilution: Begin with a 1:500 dilution in primary antibody incubation. Adjust based on signal strength in your specific experimental system.

• Detection: SuperSignal West Pico substrate or equivalent chemiluminescent detection systems provide appropriate sensitivity.

• Controls: Include positive controls (e.g., HeLa cell lysate) which have been confirmed to express detectable levels of HIST1H2BC .

For stripping and reprobing, use a gentle stripping buffer (10mM Tris-HCl [pH 2.3], 150mM NaCl) to preserve membrane integrity while enabling multiple protein analyses on the same blot .

What are the best practices for immunohistochemistry using this antibody?

For successful immunohistochemistry applications with HIST1H2BC (Ab-116) Antibody:

• Fixation: Use 4% paraformaldehyde fixation for optimal epitope preservation.

• Antigen Retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) to unmask histone epitopes that may be obscured during fixation.

• Blocking: Block with 5-10% normal serum (from the species of the secondary antibody) with 0.1-0.3% Triton X-100 for permeabilization.

• Antibody Dilution: Start with a 1:50 dilution and optimize based on your specific tissue type.

• Incubation: Overnight incubation at 4°C typically yields optimal staining results.

• Detection Systems: Both DAB-based chromogenic and fluorescence-based detection systems are compatible.

• Controls: Include both negative controls (omitting primary antibody) and positive controls (tissues known to express HIST1H2BC) .

What considerations should be made when designing immunofluorescence experiments?

For immunofluorescence applications using the HIST1H2BC (Ab-116) Antibody:

• Cell Preparation: Culture cells on coverslips or chamber slides and fix with 4% paraformaldehyde for 15 minutes.

• Permeabilization: Use 0.1-0.3% Triton X-100 in PBS for 10 minutes for nuclear protein access.

• Blocking: Block with 5% BSA or 10% normal serum with 0.1% Triton X-100 for 1 hour at room temperature.

• Antibody Dilution: Begin with the higher concentration end of the recommended range (1:1-1:10) due to the sensitivity requirements of IF.

• Nuclear Counterstaining: DAPI (1μg/mL) provides effective nuclear counterstaining to contextualize histone localization.

• Mounting: Use anti-fade mounting medium to prevent photobleaching during analysis.

• Microscopy: Confocal microscopy is preferable for detailed nuclear localization studies .

How can I use HIST1H2BC (Ab-116) Antibody to investigate epigenetic modifications in chromatin structure?

For investigating epigenetic modifications in chromatin structure using the HIST1H2BC (Ab-116) Antibody:

• Chromatin Immunoprecipitation (ChIP): The antibody can be employed in ChIP assays to identify genomic regions associated with HIST1H2BC. Standard protocol adaptations include:

  • Crosslinking cells with 1% formaldehyde for 10 minutes

  • Sonicating chromatin to 200-500bp fragments

  • Using 3-5μg antibody per ChIP reaction

  • Including appropriate IgG controls

• Co-Immunoprecipitation (Co-IP): To identify protein interactions with HIST1H2BC:

  • Use gentle lysis buffers containing 0.1-0.5% NP-40 to preserve protein-protein interactions

  • Pre-clear lysates with Protein A/G beads

  • Incubate with 2-4μg antibody overnight at 4°C

  • Analyze precipitated complexes by Western blotting for suspected interaction partners

• Sequential ChIP (Re-ChIP): For analyzing co-occupancy of HIST1H2BC with other modified histones:

  • Perform first IP with HIST1H2BC antibody

  • Elute complexes under non-denaturing conditions

  • Perform second IP with antibodies against histone modifications (e.g., H3K4me3, H3K27me3)

  • Analysis reveals genomic regions with co-localization of HIST1H2BC and specific histone marks .

What approaches can be used to study HIST1H2BC in the context of DNA damage response?

To investigate HIST1H2BC's role in DNA damage response pathways:

• DNA Damage Induction and Kinetics:

  • Treat cells with DNA damaging agents (e.g., UV, ionizing radiation, etoposide)

  • Collect time-course samples (0, 15, 30, 60, 120, 240 minutes post-treatment)

  • Analyze HIST1H2BC localization, abundance, and post-translational modifications by immunoblotting and immunofluorescence

  • Co-stain with γH2AX antibodies to correlate with DNA damage foci

• Proximity Ligation Assay (PLA):

  • Use HIST1H2BC (Ab-116) Antibody in conjunction with antibodies against DNA repair factors (e.g., 53BP1, BRCA1)

  • PLA signals indicate proximity (<40nm) between HIST1H2BC and repair factors

  • Quantify PLA signals before and after DNA damage induction

• CRISPR-Cas9 Genetic Modification:

  • Generate HIST1H2BC knockouts or specific mutations

  • Compare DNA damage response kinetics between wild-type and modified cells

  • Rescue experiments with wild-type or mutant HIST1H2BC provide functional validation .

How can I integrate HIST1H2BC analysis with transcriptional profiling?

For correlating HIST1H2BC occupancy with gene expression patterns:

• ChIP-seq and RNA-seq Integration:

  • Perform ChIP-seq using HIST1H2BC (Ab-116) Antibody

  • Conduct RNA-seq on the same cell population

  • Bioinformatic analysis to correlate HIST1H2BC occupancy with gene expression levels

  • Gene Ontology analysis of HIST1H2BC-associated genes reveals functional pathways

• Single-Cell Approaches:

  • Combine single-cell RNA-seq with single-cell CUT&Tag for HIST1H2BC

  • Identify cell sub-populations with distinct HIST1H2BC distribution patterns

  • Correlate with transcriptional heterogeneity at single-cell resolution

• Inducible Systems:

  • Establish cell lines with doxycycline-inducible HIST1H2BC expression

  • Monitor transcriptional changes upon HIST1H2BC induction using RNA-seq or qRT-PCR

  • Identify direct transcriptional targets versus secondary effects .

What are common issues when using HIST1H2BC (Ab-116) Antibody in Western blotting and how can they be resolved?

Common issues and solutions for Western blotting with HIST1H2BC (Ab-116) Antibody:

• Weak or No Signal:

  • Increase antibody concentration (try 1:100 instead of 1:1000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Ensure adequate protein loading (20-40μg total protein)

  • Verify transfer efficiency with Ponceau S staining

  • Consider alternative extraction methods optimized for nuclear proteins

• High Background:

  • Increase blocking time and concentration (5% BSA or milk for 1-2 hours)

  • Add 0.1% Tween-20 to all washing and antibody incubation steps

  • Reduce primary antibody concentration (try 1:1000 instead of 1:100)

  • Include 0.05% SDS in washing buffer for stringent washing

• Multiple Bands:

  • This may represent different histone variants or post-translational modifications

  • Use positive controls to identify the correct band (~14 kDa)

  • Consider using pre-absorbed antibody to improve specificity

  • Include appropriate blocking peptides as specificity controls .

How can I validate antibody specificity for my particular experimental system?

To ensure HIST1H2BC (Ab-116) Antibody specificity in your experimental system:

• Peptide Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare staining pattern with and without peptide competition

  • Specific signals should be abolished or significantly reduced

• Genetic Approaches:

  • Use HIST1H2BC knockdown/knockout cell lines as negative controls

  • Compare signal between wild-type and HIST1H2BC-depleted samples

  • Rescue experiments with exogenous HIST1H2BC expression provide additional validation

• Multiple Antibody Validation:

  • Use alternative HIST1H2BC antibodies targeting different epitopes

  • Concordant results with multiple antibodies increase confidence

  • Discordant results suggest potential specificity issues requiring further investigation

• Mass Spectrometry Validation:

  • Immunoprecipitate with HIST1H2BC (Ab-116) Antibody

  • Analyze precipitated proteins by LC-MS/MS

  • Confirm presence of HIST1H2BC peptides in immunoprecipitated material .

How should I optimize antibody dilutions for different experimental applications?

For optimal antibody dilution determination across applications:

• Titration Experiments:

  • For Western blotting: Test dilutions from 1:100 to 1:1000 using a consistent positive control

  • For IHC: Test dilutions from 1:10 to 1:100 on known positive tissue

  • For IF: Test dilutions from 1:1 to 1:10 on fixed cells known to express HIST1H2BC

  • For ELISA: Test dilutions from 1:2000 to 1:10000

• Signal-to-Noise Optimization:

  • Select the dilution that provides the highest specific signal with minimal background

  • Perform densitometric analysis of specific bands versus background in Western blots

  • Use ImageJ or similar software for quantitative assessment of signal-to-noise ratios

• Sample-Specific Considerations:

  • Higher antibody concentrations may be needed for formalin-fixed paraffin-embedded tissues

  • Fresh-frozen sections typically require lower antibody concentrations

  • Cell lines with high HIST1H2BC expression may allow for more dilute antibody solutions .

How can I combine HIST1H2BC analysis with other histone modification studies?

To integrate HIST1H2BC analysis with broader histone modification studies:

• Multiplexed Immunofluorescence:

  • Use spectrally distinct fluorophores for simultaneous detection of HIST1H2BC and other histone modifications

  • Apply sequential staining protocols to avoid antibody cross-reactivity

  • Employ spectral unmixing for closely overlapping fluorophores

  • Analyze co-localization at the subcellular level

• Sequential ChIP (Re-ChIP):

  • First immunoprecipitation with HIST1H2BC (Ab-116) Antibody

  • Gentle elution of protein-DNA complexes

  • Second immunoprecipitation with antibodies against specific histone modifications

  • Analysis reveals genomic regions where HIST1H2BC co-exists with specific epigenetic marks

• Mass Spectrometry-Based Approaches:

  • Isolate HIST1H2BC-containing nucleosomes using the antibody

  • Analyze associated histones for post-translational modifications by mass spectrometry

  • Quantify different modification states on histones associated with HIST1H2BC .

What considerations should be made when studying HIST1H2BC in different cell types or disease models?

For studying HIST1H2BC across different biological contexts:

• Expression Level Variation:

  • Perform preliminary Western blots to determine HIST1H2BC expression levels

  • Adjust antibody concentrations accordingly for each cell type or tissue

  • Use qRT-PCR to correlate protein levels with mRNA expression

• Disease Model Considerations:

  • In cancer models, assess HIST1H2BC distribution in relation to oncogenic pathways

  • For neurological disease models, examine HIST1H2BC in neurons versus glial cells

  • In developmental disorders, track HIST1H2BC during differentiation processes

• Species Cross-Reactivity:

  • The antibody is validated for human and rat samples

  • For mouse models, perform additional validation experiments

  • Consider sequence homology when extending to other species .

How can I use HIST1H2BC (Ab-116) Antibody in conjunction with flow cytometry and cell sorting?

For flow cytometry and cell sorting applications:

• Fixation and Permeabilization Protocol:

  • Fix cells with 2-4% paraformaldehyde for 10-15 minutes

  • Permeabilize with 90% ice-cold methanol or 0.1% Triton X-100

  • Additional permeabilization may be required for nuclear antigens like HIST1H2BC

• Staining Procedure:

  • Use higher antibody concentration (1:10 to 1:50) than for Western blotting

  • Incubate for 30-60 minutes at room temperature

  • Include appropriate isotype control at the same concentration

  • For multicolor flow, include proper compensation controls

• Analysis Considerations:

  • Gate on intact, single cells before analyzing HIST1H2BC signal

  • Consider cell cycle status when interpreting HIST1H2BC levels

  • For cell sorting, use stringent gating for high purity

  • Post-sort validation by microscopy or Western blotting confirms HIST1H2BC status .

How might HIST1H2BC (Ab-116) Antibody be integrated into single-cell epigenomic analyses?

For incorporating HIST1H2BC analysis into single-cell epigenomic workflows:

• scCUT&Tag Adaptation:

  • Optimize HIST1H2BC (Ab-116) Antibody concentration for CUT&Tag protocol

  • Employ cell barcoding strategies for multiplexed analysis

  • Integrate with single-cell RNA-seq data for correlation with gene expression

  • Apply machine learning approaches for identifying cell-type-specific patterns

• Multiplexed Epitope Detection:

  • Conjugate HIST1H2BC antibody with DNA-barcoded oligos

  • Combine with other epigenetic antibodies in CITE-seq-like approaches

  • Analyze protein-level heterogeneity alongside transcriptomic variation

  • Develop computational frameworks for multi-omic data integration

• Live-Cell Applications:

  • Consider developing Fab fragments of the antibody for live-cell imaging

  • Combine with fluorescent histone markers for dynamics studies

  • Track HIST1H2BC redistribution during cell cycle or differentiation processes .

What role might HIST1H2BC play in regulatory networks affecting B-cell development and function?

Based on current understanding of histone biology and B-cell development:

• Potential Roles in B-cell Development:

  • HIST1H2BC may interact with BAP1 deubiquitinase system, which regulates histone H2A and impacts B-cell development

  • Histone variants like HIST1H2BC could be involved in regulating immunoglobulin gene rearrangements

  • Chromatin accessibility changes during B-cell activation may involve HIST1H2BC redistribution

• Experimental Approaches:

  • ChIP-seq analysis of HIST1H2BC occupancy during B-cell development stages

  • Study HIST1H2BC distribution in germinal center B cells versus memory B cells

  • Examine HIST1H2BC occupancy at immunoglobulin loci during class switching

  • Analyze changes in HIST1H2BC post-translational modifications during B-cell activation

• Disease Relevance:

  • Investigate HIST1H2BC distribution in B-cell malignancies

  • Explore potential correlations with autoimmune disease phenotypes

  • Examine HIST1H2BC patterns in antibody-mediated immune responses .

What are the latest methodological innovations for studying histone dynamics that could be applied to HIST1H2BC research?

Cutting-edge methodologies applicable to HIST1H2BC research:

• Genomic Visualization Techniques:

  • CRISPR-based visualization systems (e.g., CRISPR-dCas9 fused to fluorescent proteins)

  • Targeted in situ sequencing for spatial genomics

  • Super-resolution microscopy for nanoscale chromatin organization

• Proximity Labeling Approaches:

  • BioID or TurboID fusion with HIST1H2BC to identify proximal proteins

  • APEX2-based labeling for temporal protein interaction mapping

  • Integration with mass spectrometry for unbiased interactome analysis

• Combinatorial Indexing Technologies:

  • sci-ATAC-seq adaptations for chromatin accessibility

  • Multimodal profiling combining chromatin state, transcription, and protein levels

  • Spatial transcriptomics integration for tissue-context epigenetic profiling

• Computational Approaches:

  • Deep learning algorithms for predicting HIST1H2BC binding sites

  • Network analysis tools for integrating HIST1H2BC with broader epigenetic mechanisms

  • Multi-scale modeling of chromatin dynamics incorporating histone variant data .