HIST1H1C (Ab-109) Antibody

What is HIST1H1C (Ab-109) Antibody and what epitope does it target?

HIST1H1C (Ab-109) Antibody is a polyclonal antibody raised in rabbits that specifically recognizes the region surrounding lysine 109 (Lys109) in human Histone H1.2 (HIST1H1C) . This histone protein, also known as H1.2, H1c, or H1d, belongs to the linker histone family that binds to DNA between nucleosomes to form higher-order chromatin structures . The antibody is generated using a synthetic peptide derived from Human Histone H1.2 protein (amino acids 100-112) . It is important to note that this antibody is specific to the unmodified form of the protein at this particular epitope, making it distinct from other HIST1H1C antibodies that target modified sites such as acetylated or methylated residues .

What applications can HIST1H1C (Ab-109) Antibody be used for?

The HIST1H1C (Ab-109) Antibody has been validated for multiple research applications:

The antibody shows strong specificity in these applications with a predicted band size of 22 kDa in Western blot analyses . For immunofluorescence, the antibody typically shows nuclear localization consistent with the chromatin-binding function of histone proteins .

What are the optimal storage and handling conditions for HIST1H1C (Ab-109) Antibody?

For maximum stability and activity retention, HIST1H1C (Ab-109) Antibody should be stored at -20°C or -80°C immediately upon receipt . The antibody is provided in liquid form with a buffer containing 50% glycerol, 0.01M PBS at pH 7.4, and 0.03% Proclin 300 as a preservative . This formulation helps maintain antibody stability during freeze-thaw cycles.

To minimize deterioration:

• Aliquot the antibody into smaller volumes to avoid repeated freeze-thaw cycles

• When thawing, allow the antibody to reach room temperature completely before use

• Briefly centrifuge the vial before opening to collect all liquid at the bottom

• Return to -20°C or -80°C immediately after use

• Avoid more than 5 freeze-thaw cycles which can compromise antibody performance

How does HIST1H1C function in chromatin organization and gene regulation?

HIST1H1C (Histone H1.2) plays crucial roles in chromatin organization and gene regulation through several mechanisms:

• Chromatin Structure Formation: HIST1H1C binds to linker DNA between nucleosomes, forming the macromolecular structure known as chromatin fiber. It is necessary for the condensation of nucleosome chains into higher-order structured fibers .

• Transcriptional Regulation: HIST1H1C functions as a regulator of individual gene transcription through multiple mechanisms:

  • Chromatin remodeling

  • Nucleosome spacing

  • DNA methylation (by similarity)

• Immune Response Modulation: Research has shown that HIST1H1C regulates interferon-β (IFN-β) production and inhibits influenza virus replication . The C-terminal domain of H1C interacts with NS2 protein of influenza virus in the nucleus, affecting viral replication efficiency .

• Post-translational Modification Sites: HIST1H1C contains multiple residues that undergo various modifications:

  • Lysine 34 (K34) and Lysine 187 (K187): Methylation sites that enhance IFN-β production

  • Threonine 146 (T146): Phosphorylation site that decreases IFN-β production

  • Lysine 62 (K62): Acetylation site with regulatory functions

These modifications can significantly alter the protein's function in gene expression and cellular response to pathogens.

How can HIST1H1C (Ab-109) Antibody be used to investigate protein-protein interactions?

HIST1H1C (Ab-109) Antibody can be effectively used in co-immunoprecipitation (Co-IP) experiments to study protein-protein interactions involving HIST1H1C. Research findings demonstrate a methodological approach:

• Cell Preparation:

  • Transfect HEK293T cells with tagged versions of HIST1H1C and potential interacting proteins (e.g., HA-IRF3 and Flag-H1C)

  • Infect cells with virus or stimulate with appropriate factors if studying interaction under specific conditions

  • Harvest cells at appropriate timepoints (typically 10-24 hours post-treatment)

• Co-IP Protocol:

  • Lyse cells with appropriate buffer

  • Perform immunoprecipitation using HIST1H1C (Ab-109) Antibody at 1:50 - 1:100 dilution

  • Incubate with protein A/G beads

  • Wash extensively to remove non-specific interactions

  • Elute protein complexes and analyze by Western blotting

• Detection of Interactions:

  • Published studies have used this approach to identify interactions between HIST1H1C and:

    • Influenza virus NS2 protein

    • IRF3 (Interferon Regulatory Factor 3)

    • Various nuclear proteins involved in transcriptional regulation

This method has revealed that the C-terminal domain of HIST1H1C mediates interaction with NS2, while different regions may be involved in IRF3 binding .

What controls should be included when using HIST1H1C (Ab-109) Antibody?

To ensure experimental validity when using HIST1H1C (Ab-109) Antibody, researchers should incorporate the following controls:

• Positive Controls:

  • Cell lines known to express HIST1H1C (validated options include HeLa, A549, and HL60 cells)

  • Recombinant HIST1H1C protein as a standard for Western blot

  • Nuclear extracts from human cell lines (as HIST1H1C is predominantly nuclear)

• Negative Controls:

  • HIST1H1C knockout cell lines (A549-H1C-KO cells have been generated using CRISPR/Cas9)

  • Cells where HIST1H1C has been silenced using siRNA

  • Secondary antibody-only controls for immunofluorescence and IHC to assess background

• Specificity Controls:

  • Pre-incubation of antibody with immunizing peptide to block specific binding

  • Use of other H1 histone variant antibodies to confirm isoform specificity (e.g., testing cross-reactivity with H1.0)

  • Use of loading controls in Western blot (e.g., alpha-tubulin antibody as demonstrated in validation studies)

• Technical Controls:

  • IgG isotype control for immunoprecipitation experiments

  • Subcellular fractionation controls to verify nuclear localization

  • Blocking peptide competition assays to confirm specificity

Validation studies have demonstrated that this antibody shows loss of signal in HIST1H1C knockout samples, confirming its specificity .

How can HIST1H1C (Ab-109) Antibody be used to study the role of HIST1H1C in interferon regulation and antiviral responses?

HIST1H1C plays a significant role in interferon-β (IFN-β) regulation and antiviral responses. The HIST1H1C (Ab-109) Antibody can be employed in several sophisticated experimental approaches to investigate these functions:

• Chromatin Immunoprecipitation (ChIP) Analysis:

  • Use HIST1H1C (Ab-109) Antibody (1:100 dilution) to immunoprecipitate chromatin

  • Analyze the association of HIST1H1C with IFN-β promoter regions

  • Compare binding patterns in virus-infected versus uninfected cells

  • Correlate HIST1H1C occupancy with transcriptional activation

• Mutational Analysis Combined with Antibody Detection:

  • Generate HIST1H1C mutants at key modification sites (K34A, K187A, T146A)

  • Transfect cells with these mutants and detect expression using the antibody

  • Quantify IFN-β production through qRT-PCR and compare with wild-type HIST1H1C

  • Results have shown that K34A and K187A mutations enhance IFN-β production, while T146A decreases it

• Co-immunoprecipitation of Transcription Factors:

  • Use HIST1H1C (Ab-109) Antibody to pull down HIST1H1C protein complexes

  • Analyze interaction with transcription factors like IRF3

  • Research has shown that HIST1H1C interacts with IRF3, and this interaction is affected by viral proteins like NS2

• Virus Replication Assays:

  • Manipulate HIST1H1C expression (overexpression, silencing, or knockout)

  • Infect cells with influenza virus

  • Measure virus titers and correlate with HIST1H1C levels

  • Research has demonstrated that virus proliferates more robustly in A549-H1C-KO cells compared to wild-type cells

This methodological approach has revealed that HIST1H1C inhibits influenza virus replication primarily by upregulating IFN-β production, and this function is modulated by specific post-translational modifications at K34, K187, and T146 residues .

What methodologies can be used to study HIST1H1C post-translational modifications using HIST1H1C antibodies?

Studying post-translational modifications (PTMs) of HIST1H1C requires specialized methodologies that can be implemented using both HIST1H1C (Ab-109) Antibody and modification-specific antibodies:

• Sequential Immunoprecipitation Approach:

  • First IP: Use modification-specific antibodies (e.g., anti-acetyl-Lys62 HIST1H1C)

  • Second IP: Re-immunoprecipitate with HIST1H1C (Ab-109) Antibody

  • This approach identifies the proportion of HIST1H1C with specific modifications

• Mass Spectrometry Analysis of Immunoprecipitated Samples:

  • Immunoprecipitate HIST1H1C using HIST1H1C (Ab-109) Antibody

  • Perform tryptic digestion of purified protein

  • Analyze peptides by LC-MS/MS

  • Identify and quantify various PTMs including:

    • Acetylation (e.g., K16, K62, K84, K96)

    • Methylation (e.g., K45, K96, K186)

    • Phosphorylation (e.g., T146)

• Site-Directed Mutagenesis Combined with Functional Assays:

  • Generate HIST1H1C mutants at specific modification sites

  • Express mutants in HIST1H1C-knockout cells

  • Use HIST1H1C (Ab-109) Antibody to confirm expression

  • Assess functional consequences through:

    • IFN-β production

    • Virus replication assays

    • Chromatin binding studies

• Immunofluorescence Colocalization Studies:

  • Perform double immunofluorescence with:

    • HIST1H1C (Ab-109) Antibody (1:10-1:100 dilution)

    • Modification-specific antibodies

  • Analyze colocalization patterns

  • Assess changes in response to cellular stimuli or pathogen infection

Research using these approaches has demonstrated that different modifications of HIST1H1C modulate its function in immune responses, with methylation at K34 and K187 enhancing IFN-β production and phosphorylation at T146 inhibiting it .

How can HIST1H1C (Ab-109) Antibody be used to investigate autophagy in disease models?

Recent research has identified a role for HIST1H1C in regulating autophagy, particularly in the context of diabetic retinopathy. HIST1H1C (Ab-109) Antibody can be employed in several methodological approaches to investigate this function:

• In Vivo and In Vitro Expression Analysis:

  • Immunohistochemistry (IHC) using HIST1H1C (Ab-109) Antibody (1:20-1:200 dilution) in retinal tissues from diabetic models

  • Western blot analysis (1:500-1:2000 dilution) to quantify HIST1H1C expression in:

    • Diabetic retina samples

    • High glucose-treated retinal cells

• Autophagy Pathway Investigation:

  • Co-transfection experiments with:

    • HIST1H1C expression plasmids (pH1.2)

    • GFP-LC3 (autophagy marker)

  • Visualization of autophagy using confocal microscopy

  • Quantification of autophagic cells (defined as cells containing >10 cytoplasmic GFP dots)

  • Validation with HIST1H1C (Ab-109) Antibody to confirm expression

• Autophagy Flux Assessment:

  • HIST1H1C overexpression or knockdown in cell models

  • Treatment with or without autophagy inhibitors (chloroquine, bafilomycin A1)

  • Western blot analysis of autophagy markers (LC3-II, p62)

  • Correlation with HIST1H1C expression levels

• Stable Knockdown Cell Line Validation:

  • Generate stable HIST1H1C knockdown cell lines using shRNA

  • Validate knockdown efficiency using HIST1H1C (Ab-109) Antibody

  • Compare autophagy markers and response to stress conditions

  • Assess potential protective or pathological effects

Studies have found that both HIST1H1C and autophagy-related proteins (ATG) are upregulated in the retinas of diabetic models, suggesting a potential therapeutic target for diabetic retinopathy .

What are the methodological considerations for using HIST1H1C (Ab-109) Antibody in subcellular fractionation experiments?

When using HIST1H1C (Ab-109) Antibody for subcellular fractionation analysis, researchers should consider the following methodological approach:

• Fractionation Protocol Optimization:

  • Extract subcellular fractions from HEK293T or A549 cells following established protocols:

    • Lyse cells with cytoplasmic extraction buffer (on ice for 20 min)

    • Add NP-40 to 0.5% final concentration and vortex briefly

    • Centrifuge at 3,500g for 10 min at 4°C

    • Collect supernatant as cytoplasmic fraction

    • Dissolve pellet in nuclear extraction buffer (10 min on ice)

    • Centrifuge at 14,000g for 10 min at 4°C

    • Collect supernatant as nuclear fraction

• Western Blot Analysis of Fractions:

  • Use HIST1H1C (Ab-109) Antibody at 1:500-1:2000 dilution

  • Include appropriate fraction markers:

    • Nuclear fraction: Lamin B1 or Histone H3

    • Cytoplasmic fraction: α-tubulin or GAPDH

  • Quantify relative distribution of HIST1H1C between compartments

  • Assess changes in localization in response to stimuli or infection

• Immunofluorescence Validation:

  • Perform immunofluorescence using HIST1H1C (Ab-109) Antibody (1:10-1:100)

  • Counterstain with DAPI for nuclear visualization

  • Analyze colocalization with other nuclear markers

  • Confirm predominant nuclear localization under normal conditions

• Experimental Conditions to Consider:

  • Viral infection: May alter HIST1H1C localization

  • Cell cycle phase: Distribution may vary during different cell cycle stages

  • Post-translational modifications: Can affect nuclear retention or export

  • Fixation method for immunofluorescence: Critical for preserving nuclear architecture

Research has shown that HIST1H1C predominantly localizes to the nucleus, where it interacts with NS2 protein of influenza virus via its C-terminal domain. This interaction in the nucleus affects viral replication and host immune responses .

How can I troubleshoot non-specific binding or weak signals when using HIST1H1C (Ab-109) Antibody?

When experiencing issues with HIST1H1C (Ab-109) Antibody performance, consider the following troubleshooting approaches:

For Western blot applications specifically, successful detection of HIST1H1C has been demonstrated in HeLa, A549, and HL60 whole cell lysates with a predicted band size of 22 kDa using the antibody at 1:500 dilution followed by HRP-conjugated secondary antibody .

What experimental design considerations should be made when studying HIST1H1C expression in different cell types?

When designing experiments to study HIST1H1C expression across different cell types, consider the following methodological approaches:

• Cell Type Selection and Preparation:

  • Choose relevant cell types based on research question:

    • Cancer cell lines: HeLa, A549, HL60 (validated for antibody)

    • Primary cells: Consider tissue-specific expression patterns

    • Diseased vs. normal tissues: Important for pathological studies

  • Standardize cell culture conditions to minimize variability

  • Control for cell cycle stage, as histone expression may vary during cell cycle

• Expression Analysis Methodology:

  • Western Blot:

    • Standardize protein extraction (consider nuclear extraction protocols)

    • Load equal amounts of protein (10-30 μg per lane)

    • Include loading controls appropriate for nuclear proteins

    • Use HIST1H1C (Ab-109) Antibody at 1:500-1:2000 dilution

  • Immunofluorescence:

    • Optimize fixation method (4% formaldehyde recommended)

    • Permeabilize with 0.2% Triton X-100

    • Block with 10% normal goat serum

    • Use HIST1H1C (Ab-109) Antibody at 1:10-1:100 dilution

    • Counterstain with DAPI for nuclear visualization

• Quantification Approaches:

  • For Western blot: Densitometric analysis normalized to loading controls

  • For immunofluorescence: Measure nuclear fluorescence intensity

  • For IHC: Consider H-score or other semi-quantitative scoring methods

  • Use appropriate statistical tests for comparisons between cell types

• Validation Strategies:

  • Include multiple detection methods (WB, IF, IHC)

  • Validate with orthogonal approaches (qRT-PCR for mRNA)

  • Consider knockout/knockdown validation

  • Include positive controls (cell types known to express HIST1H1C)

Research has demonstrated that HIST1H1C expression may vary in different physiological and pathological conditions, and can be regulated in response to stimuli like viral infection .

How is HIST1H1C (Ab-109) Antibody being used in current epigenetic research?

HIST1H1C (Ab-109) Antibody has become an important tool in epigenetic research, particularly in studies investigating chromatin structure regulation and gene expression. Current methodological applications include:

• Chromatin Immunoprecipitation Sequencing (ChIP-seq):

  • Use HIST1H1C (Ab-109) Antibody to immunoprecipitate chromatin

  • Sequence associated DNA to identify genome-wide binding patterns

  • Compare HIST1H1C occupancy with:

    • Active vs. repressed chromatin regions

    • DNA methylation patterns

    • Other histone modifications (H3K4me3, H3K27me3, etc.)

  • Correlate binding patterns with transcriptional outcomes

• Post-translational Modification (PTM) Crosstalk Analysis:

  • Compare chromatin immunoprecipitated with:

    • HIST1H1C (Ab-109) Antibody (unmodified form)

    • Modification-specific antibodies (acLys62, meLys45, etc.)

  • Investigate how different modifications affect chromatin binding and gene regulation

  • Explore the "histone code" in the context of linker histones

• Nucleosome Positioning Studies:

  • Combine HIST1H1C ChIP with nucleosome mapping techniques

  • Analyze how HIST1H1C binding affects:

    • Nucleosome repeat length

    • Chromatin accessibility

    • Higher-order chromatin structure

  • Correlate with gene expression patterns

• Disease-Associated Epigenetic Alterations:

  • Compare HIST1H1C binding patterns in:

    • Normal vs. disease tissues

    • Response to treatments

    • Various developmental stages

  • Identify potential epigenetic biomarkers

  • Explore therapeutic targets for epigenetic manipulation

Current research has revealed that HIST1H1C plays important roles beyond structural chromatin organization, including specific gene regulation and immune response modulation through its involvement in interferon pathways .

What is the role of HIST1H1C in disease pathogenesis and how can HIST1H1C (Ab-109) Antibody contribute to these studies?

HIST1H1C has emerged as a significant factor in various disease processes, and HIST1H1C (Ab-109) Antibody provides valuable methodological approaches for investigating these pathological roles:

• Viral Infection and Immune Response:

  • HIST1H1C regulates interferon-β (IFN-β) production and inhibits influenza virus replication

  • Methodological approach:

    • Use HIST1H1C (Ab-109) Antibody to track expression during infection

    • Compare virus replication in wild-type vs. HIST1H1C knockout cells

    • Analyze interaction with viral proteins (NS2) via co-immunoprecipitation

    • Measure cytokine production (IFN-β, TNF-α, CXCL10) in relation to HIST1H1C levels

  • Research has shown that influenza virus replicates better in H1C knockout cells due to reduced IFN-β production

• Diabetic Retinopathy and Autophagy Regulation:

  • HIST1H1C and autophagy-related proteins are upregulated in diabetic retinas

  • Methodological approach:

    • Use HIST1H1C (Ab-109) Antibody to detect expression in retinal tissues

    • Correlate with autophagy markers (LC3-II, p62)

    • Analyze effects of HIST1H1C manipulation on autophagy flux

    • Investigate potential therapeutic targeting of this pathway

  • Studies indicate that HIST1H1C may play a role in the development of diabetic retinopathy through autophagy regulation

• Cancer Research Applications:

  • HIST1H1C may be involved in chromatin remodeling and gene expression in cancer cells

  • Methodological approach:

    • Use HIST1H1C (Ab-109) Antibody in immunohistochemistry of cancer tissues

    • Analyze correlation with clinical parameters and prognosis

    • Investigate epigenetic alterations and their functional consequences

    • Explore potential as a biomarker or therapeutic target

• Neurodegenerative Disorders:

  • Emerging evidence suggests histone modifications may contribute to neurodegenerative processes

  • Methodological approach:

    • Use HIST1H1C (Ab-109) Antibody to analyze expression in neural tissues

    • Investigate changes in histone modifications in disease models

    • Correlate with transcriptional alterations of key pathological genes

These methodological approaches demonstrate how HIST1H1C (Ab-109) Antibody can contribute to understanding disease mechanisms and potentially identifying novel therapeutic targets.