HIST1H1C (Ab-45) Antibody

What is HIST1H1C and what is its biological significance?

Answer: HIST1H1C (also known as Histone H1.2) is a linker histone protein that binds to DNA between nucleosomes, forming the macromolecular structure known as chromatin fiber. It plays essential roles in chromatin organization by facilitating the condensation of nucleosome chains into higher-order structured fibers. Beyond its structural function, HIST1H1C acts as a regulator of gene transcription through multiple mechanisms including chromatin remodeling, nucleosome spacing, and DNA methylation . Recent research has revealed that HIST1H1C is involved in coupling cellular mechanical behaviors to chromatin regulation, affecting contractile force generation, cytoskeletal regulation, cell motility, and extracellular matrix deposition .

What applications is the HIST1H1C (Ab-45) antibody validated for?

Answer: The HIST1H1C (Ab-45) polyclonal antibody has been validated for multiple research applications, including:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB)

• Immunohistochemistry (IHC)

• Chromatin Immunoprecipitation (ChIP)

• Immunofluorescence (IF) in some versions

The recommended dilutions vary by application: for Western Blotting, typically 1:100-1:1000; for IHC, 1:10-1:100 . These applications enable researchers to investigate HIST1H1C expression, localization, and interaction with chromatin in various experimental contexts.

What are the storage and handling requirements for HIST1H1C (Ab-45) antibody?

Answer: For optimal antibody performance, store HIST1H1C (Ab-45) antibody at -20°C or -80°C immediately upon receipt. The antibody is provided in liquid form in a buffer containing 0.03% Proclin 300 as a preservative, 50% glycerol, and 0.01M PBS at pH 7.4 . Critical to maintaining antibody integrity is avoiding repeated freeze-thaw cycles, which can cause protein denaturation and loss of binding activity. When working with the antibody, aliquot into smaller volumes for single use if conducting multiple experiments over time. For short-term storage during experimentation, keep on ice and return to appropriate freezer promptly after use .

How should I design ChIP experiments using HIST1H1C (Ab-45) antibody?

Answer: When designing Chromatin Immunoprecipitation (ChIP) experiments with HIST1H1C (Ab-45) antibody, follow these methodological guidelines:

• Crosslinking: Fix cells with 1% formaldehyde for 10 minutes at room temperature to preserve protein-DNA interactions.

• Chromatin preparation: After cell lysis, sonicate chromatin to achieve fragment sizes of 200-500 bp, which is optimal for histone studies.

• Immunoprecipitation: Use 2-5 μg of HIST1H1C (Ab-45) antibody per ChIP reaction with 25-50 μg of chromatin. Include an IgG control to assess background.

• Washing and elution: Perform stringent washes to remove non-specific binding before eluting DNA-protein complexes.

• Analysis methods: For genome-wide distribution studies, combine ChIP with sequencing (ChIP-seq) or tiling arrays as demonstrated in breast cancer cells .

Recent research has successfully used this antibody to map the distribution of histone variants in breast cancer cells through ChIP combined with qPCR and high-resolution sequencing, revealing that H1 variants show specific distribution patterns across the genome .

What is the optimal protocol for immunofluorescence staining with HIST1H1C (Ab-45) antibody?

Answer: For optimal immunofluorescence staining with HIST1H1C (Ab-45) antibody, follow this protocol adapted from recent research:

• Cell preparation: Grow cells directly on high-performance glass coverslips (0.17 mm thickness).

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde for 20 minutes at room temperature

  • Permeabilize with methanol for 10 minutes at room temperature

  • Block with 5% bovine serum albumin (BSA) in PBS with 0.1% Triton X-100

• Antibody incubation:

  • Incubate with HIST1H1C (Ab-45) antibody at 1:100 dilution overnight at 4°C

  • Wash three times with PBS-T (PBS with 0.1% Triton X-100)

  • Incubate with appropriate secondary antibody (e.g., goat anti-rabbit IgG conjugated with Alexa-488 or -647) for 1 hour at room temperature in the dark

• Nuclear counterstaining:

  • Stain nuclei with Hoechst (25 μg/ml) for 1 hour at room temperature

  • Wash five times with PBS-T and once with Milli-Q water

• Mounting and imaging:

  • Mount using Prolong Glass mounting medium

  • Allow slides to cure 24-48 hours at room temperature before imaging

For studying mitotic distribution of HIST1H1C, synchronize cells using Thymidine-Nocodazole treatment to increase the percentage of mitotic cells .

How can I validate the specificity of HIST1H1C (Ab-45) antibody in my experiments?

Answer: Validating antibody specificity is critical for reliable experimental results. For HIST1H1C (Ab-45) antibody, implement these validation approaches:

• Positive and negative controls:

  • Positive control: Use cell lines known to express HIST1H1C (such as T47D breast cancer cells)

  • Negative control: Include samples where HIST1H1C expression is knocked down via siRNA or shRNA

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (sequence around Lysine 45 of Histone H1.2) before application to samples. Signal reduction confirms specificity.

• Knockdown validation: Establish stable HIST1H1C knockdown cell lines using shRNA or siRNA targeting, as demonstrated in rMC-1 cells, and confirm reduced signal by Western blot and qPCR .

• Cross-reactivity assessment: Test on samples from different species to confirm the antibody's reactivity with mouse and rat HIST1H1C, as claimed in the product specifications .

• Multiple detection methods: Confirm results using at least two techniques (e.g., Western blot and immunofluorescence) to ensure consistent detection patterns.

In published research, specificity was confirmed by detecting significant decreases in both HIST1H1C mRNA and protein levels in sh-HIST1H1C rMC-1 cells compared to control cells .

How does HIST1H1C distribution vary across the genome and what does this indicate?

Answer: Recent high-resolution mapping studies of HIST1H1C distribution across the genome have revealed several key patterns that inform our understanding of its functional roles:

• Chromosomal distribution: HIST1H1C shows differential occupancy across human chromosomes, with variation correlating with both gene expression levels and gene richness coefficient (GRC) of each chromosome .

• Correlation with chromatin states: HIST1H1C distribution shows:

  • Negative correlation with H3K4me3 (active promoter mark)

  • Positive correlation with H3K9me3 (heterochromatin mark)

  • Depletion from GC-rich and gene-rich regions and active promoters

  • Overrepresentation in major satellites

• Cell-type specific patterns: In differentiated cells (like IMR90 fibroblasts), HIST1H1C shows enrichment in zones of gene repression and chromatin compaction, which is not observed in embryonic stem cells .

• Functional implications: The heterogeneous distribution pattern suggests HIST1H1C plays specific roles in chromatin regulation rather than serving merely as a structural protein. This contributes to the emerging understanding that histone H1 variants have distinct functions despite their structural similarities .

These findings can be visualized by generating heat maps and dendrograms of occupancy data across chromosomes, combined with correlation analyses between HIST1H1C occupancy, gene expression, and gene richness coefficient .

What role does HIST1H1C play in regulating autophagy and how can this be studied?

Answer: HIST1H1C has been identified as a critical regulator of autophagy, particularly in the context of diabetic retinopathy. To study this relationship, researchers can employ several methodological approaches:

• Autophagy assessment after HIST1H1C manipulation:

  • Overexpress HIST1H1C via transfection (e.g., using pH1.2 plasmid)

  • Assess autophagy by:

    • Measuring conversion of LC3B-I to LC3B-II by Western blot

    • Co-transfecting GFP-LC3 to visualize autophagy (cells with >10 cytoplasmic GFP dots are considered autophagic)

    • Quantifying SQSTM1/p62 levels (a substrate of autophagy)

• Autophagy flux assessment:

  • Treat HIST1H1C-overexpressing cells with autophagy inhibitors (chloroquine at 50μM or bafilomycin A1 at 100nM for 12h)

  • Monitor SQSTM1 accumulation and further LC3B conversion, which indicates enhanced autophagy flux

• Stress response studies:

  • Expose HIST1H1C knockdown cells to autophagy inducers (starvation, rapamycin, high glucose)

  • Quantify autophagy markers to determine if HIST1H1C is necessary for stress-induced autophagy

Research has shown that HIST1H1C overexpression upregulates SIRT1 and HDAC1 to maintain H4K16 deacetylation status, leading to upregulation of ATG proteins (ATG12-ATG5 complex, ATG7, ATG3) and enhanced autophagy. Conversely, knockdown of HIST1H1C reduces both basal and stress-induced autophagy .

How can I interpret conflicting results between HIST1H1C localization and function in different cell types?

Answer: When facing conflicting results regarding HIST1H1C localization and function across different cell types, consider these analytical approaches:

• Cell type-specific context:

  • Different cell types may express varying levels of HIST1H1C interaction partners or chromatin modifiers

  • Compare your findings with published data on tissue and cell-type specific expression patterns of H1 variants

• Subcellular localization analysis:

  • Perform nuclear/cytoplasmic fractionation assays to determine exact localization

  • Use high-resolution imaging (confocal microscopy) with appropriate controls

  • Consider that translocation events may be time-dependent or stimulus-specific

• Functional redundancy assessment:

  • Some studies suggest compensatory upregulation of other H1 variants when one is depleted

  • Examine expression levels of all H1 variants (H1.0-H1.5, H1X) when manipulating HIST1H1C

• Chromatin state considerations:

  • Cell-type specific chromatin landscapes may alter HIST1H1C binding patterns

  • Evaluate modifications like DNA methylation (using 5-aza-2'-deoxycytidine treatment at 5μM for 3 days)

  • Consider transcriptional activity (using ActD at 50ng/ml for 24hr)

Research has shown that while HIST1H1C was previously reported to function as an apoptosis mediator when translocated to the cytoplasm upon X-ray irradiation, it remains predominantly nuclear in retinal cells under high glucose conditions, suggesting context-dependent functions . This demonstrates that HIST1H1C may have multiple distinct roles depending on cellular context and experimental conditions.

How can HIST1H1C (Ab-45) antibody be used to study the relationship between histone modifications and HIST1H1C binding?

Answer: To investigate the relationship between histone modifications and HIST1H1C binding, implement these advanced methodological approaches:

• Sequential ChIP (Re-ChIP):

  • Perform first ChIP with HIST1H1C (Ab-45) antibody

  • Elute the immunoprecipitated complexes

  • Perform second ChIP with antibodies against specific histone modifications (e.g., H3K27Ac, H3K9me3, H4K16Ac)

  • This reveals genomic regions where HIST1H1C co-occurs with specific modifications

• ChIP-seq with differential modification analysis:

  • Generate genome-wide maps of HIST1H1C binding using ChIP-seq

  • Compare with published datasets of histone modifications

  • Identify correlations between HIST1H1C occupancy and specific modification patterns

  • Research has shown negative correlation with H3K4me3 and positive correlation with H3K9me3

• Manipulation of histone-modifying enzymes:

  • Overexpress or inhibit histone deacetylases (HDACs) or methyltransferases

  • Compare HIST1H1C binding patterns before and after manipulation

  • Studies have shown HIST1H1C upregulates SIRT1 and HDAC1 to maintain H4K16 deacetylation status

• In vitro binding assays with modified nucleosomes:

  • Prepare reconstituted nucleosomes with specific histone modifications

  • Assess HIST1H1C binding affinity using purified protein

  • Compare binding to modified vs. unmodified nucleosomes

Recent findings indicate that histone H1.0 (related to HIST1H1C) is required for cytokine-induced reprogramming of H3K27Ac and acts via modulation of HDACs and BRD4 , suggesting complex interplay between linker histones and core histone modifications.

What approaches can be used to study HIST1H1C's role in cellular mechanical behaviors and cytoskeletal regulation?

Answer: To investigate HIST1H1C's emerging role in cellular mechanical behaviors and cytoskeletal regulation, employ these methodological approaches:

• Traction force microscopy:

  • Culture cells with manipulated HIST1H1C levels on polyacrylamide gels embedded with fluorescent beads

  • Measure gel deformations to quantify contractile forces

  • Compare force generation between control, HIST1H1C-overexpressing, and HIST1H1C-knockdown cells

• Live-cell cytoskeletal imaging:

  • Transfect cells with fluorescently tagged cytoskeletal proteins (actin, tubulin)

  • Perform time-lapse imaging in cells with altered HIST1H1C expression

  • Analyze dynamics and organization of cytoskeletal networks

• Cell migration assays:

  • Conduct wound healing or transwell migration assays

  • Track individual cell movements using time-lapse microscopy

  • Quantify migration parameters (velocity, directionality, persistence)

• Extracellular matrix deposition analysis:

  • Measure ECM protein production (e.g., collagen, fibronectin) via immunofluorescence or ELISA

  • Analyze matrix organization using techniques like second harmonic generation microscopy

  • Compare ECM characteristics between control and HIST1H1C-manipulated cells

• Correlation with chromatin accessibility:

  • Combine mechanical studies with ATAC-seq to assess chromatin accessibility

  • Identify mechanosensitive genes regulated by HIST1H1C

Recent research has demonstrated that HIST1H1C affects a wide range of mechanical behaviors in cells, including contractile force generation, cytoskeletal regulation, motility, and ECM deposition through its chromatin regulatory actions .

How can advanced imaging techniques be combined with HIST1H1C (Ab-45) antibody for studying chromatin dynamics?

Answer: Combining advanced imaging techniques with HIST1H1C (Ab-45) antibody enables sophisticated analysis of chromatin dynamics. Implement these methodological approaches:

• Super-resolution microscopy:

  • Use techniques like STED, STORM, or PALM with HIST1H1C (Ab-45) antibody

  • Achieve resolution below the diffraction limit (~20-50 nm)

  • Visualize precise distribution of HIST1H1C within chromatin domains

  • Protocol modification: Use appropriate fluorophore-conjugated secondary antibodies optimized for super-resolution (e.g., Alexa-647)

• Live-cell imaging of chromatin dynamics:

  • Generate cell lines expressing fluorescently tagged HIST1H1C

  • Perform simultaneous imaging with markers of chromatin states

  • Track changes in HIST1H1C distribution during cell cycle progression

  • For mitotic studies, synchronize cells using Thymidine-Nocodazole treatment

• FRAP (Fluorescence Recovery After Photobleaching):

  • Apply to cells expressing fluorescently tagged HIST1H1C

  • Measure recovery kinetics to determine binding/unbinding rates to chromatin

  • Compare dynamics in different chromatin regions (heterochromatin vs. euchromatin)

• Proximity ligation assay (PLA):

  • Combine HIST1H1C (Ab-45) antibody with antibodies against other chromatin factors

  • Detect protein-protein interactions within 40 nm distance

  • Visualize and quantify interactions in different nuclear domains

• Correlative light and electron microscopy (CLEM):

  • Locate HIST1H1C by fluorescence microscopy

  • Examine the same area by electron microscopy

  • Correlate HIST1H1C distribution with ultrastructural features of chromatin

Recent research using imaging analysis of histone H1 variants has revealed universal binding of all variants to chromatin but with distinct distribution patterns across the genome, suggesting functional specialization .

What are the common challenges when using HIST1H1C (Ab-45) antibody in ChIP experiments and how can they be addressed?

Answer: When performing ChIP experiments with HIST1H1C (Ab-45) antibody, researchers may encounter several challenges that can be systematically addressed:

• Low or variable immunoprecipitation efficiency:

  • Optimize chromatin fragmentation to 200-500 bp fragments

  • Increase antibody amount to 3-5 μg per reaction

  • Extend incubation time to overnight at 4°C with gentle rotation

  • Pre-clear chromatin with protein A/G beads to reduce background

• High background signal:

  • Include more stringent wash steps (increase salt concentration in wash buffers)

  • Add a competing protein (BSA) to block non-specific binding

  • Always perform parallel IgG control to determine background levels

  • Consider pre-absorbing the antibody with chromatin from knockdown cells

• Cross-reactivity with other H1 variants:

  • Validate specificity through Western blot of H1 variant knockdown cells

  • Perform peptide competition assays with the immunizing peptide

  • Compare ChIP-seq profiles with other H1 variant antibodies to identify unique vs. overlapping signals

• Poor enrichment at expected genomic regions:

  • Optimize fixation time (8-12 minutes usually optimal for histones)

  • Test sonication conditions to ensure accessibility of epitopes

  • Confirm target expression in your specific cell type

  • Consider epitope masking due to chromatin compaction or protein interactions

Research involving ChIP with HIST1H1C antibody in breast cancer cells successfully mapped distribution patterns by combining ChIP with quantitative PCR, tiling promoter arrays, and high-resolution sequencing, demonstrating these challenges can be overcome with proper optimization .

How can I troubleshoot inconsistent results between Western blotting and immunofluorescence when using HIST1H1C (Ab-45) antibody?

Answer: When facing discrepancies between Western blotting and immunofluorescence results with HIST1H1C (Ab-45) antibody, implement this systematic troubleshooting approach:

• Sample preparation differences:

  • Western blotting uses denatured proteins, while IF detects native conformation

  • For Western blotting, ensure complete nuclear protein extraction with specialized buffers containing high salt concentration (≥300mM NaCl)

  • For IF, test different fixation methods (4% PFA for 20 min followed by methanol for 10 min has proven effective)

• Epitope accessibility issues:

  • In IF, test different permeabilization methods (Triton X-100, methanol, or combination)

  • For Western blotting, ensure complete denaturation (boil samples for 5-10 minutes with SDS-containing buffer)

  • Consider antigen retrieval methods for IF (heat or enzymatic methods)

• Antibody concentration optimization:

  • For Western blotting: Test dilution series (1:100 to 1:1000)

  • For IF: Generally more concentrated antibody is needed (1:10 to 1:100)

  • Extend primary antibody incubation to overnight at 4°C for both methods

• Cross-validation approaches:

  • Perform nuclear/cytoplasmic fractionation assay to confirm subcellular localization

  • Use HIST1H1C knockdown or overexpression controls in both applications

  • Try alternative antibodies against HIST1H1C from different sources

• Detection system sensitivity:

  • For Western blotting, try enhanced chemiluminescence (ECL) or fluorescent secondary antibodies

  • For IF, use appropriate high-sensitivity fluorophores (Alexa-488 or -647) and optimize exposure settings

Research has shown that HIST1H1C is predominantly nuclear in both normal and high glucose conditions, which can be confirmed using both immunofluorescence staining and nuclear/cytoplasmic fractionation assays .

What are the key applications and performance characteristics of HIST1H1C (Ab-45) antibody?

Table compiled from product specifications and published research applications

How does HIST1H1C overexpression affect autophagy and inflammatory markers?

Data derived from research on rMC-1 retinal cells with overexpression of HIST1H1C

How do different H1 variants distribute across human chromosomes?

Table represents general trends based on ChIP-seq data analysis of HIST1H1C distribution across human chromosomes in breast cancer cells. Occupancy is presented as relative levels based on input-subtracted ChIP-seq signal in 50bp windows .