Formyl-HIST1H1C (K84) Antibody

What is Formyl-HIST1H1C (K84) Antibody and what does it detect?

Formyl-HIST1H1C (K84) Antibody is a polyclonal antibody that specifically recognizes the formylated lysine at position 84 of human Histone H1.2 (HIST1H1C). This antibody is designed to detect post-translational modifications (PTMs) on histone proteins that may have significant regulatory functions in chromatin structure and gene expression. The antibody (catalog number orb417591) is raised in rabbits against a peptide sequence surrounding the formyl-Lys (84) site derived from Human Histone H1.2 . This specific formylation is an important epigenetic marker that may regulate chromatin accessibility and gene expression patterns.

How does HIST1H1C differ from other histone variants?

HIST1H1C (Histone H1.2) is one of several H1 histone variants that functions as a linker histone. Unlike core histones (H2A, H2B, H3, and H4) that form nucleosome octamers, H1 histones bind to the nucleosome at the entry and exit sites of DNA, facilitating higher-order chromatin structure. HIST1H1C has a calculated molecular weight of 21 kDa, though it typically runs at 32-33 kDa on SDS-PAGE gels due to its highly basic nature and post-translational modifications . It is distinguished from other H1 variants by its specific amino acid sequence, temporal expression patterns during the cell cycle, and tissue distribution. Research indicates HIST1H1C may have specialized functions in certain cellular contexts, particularly in nuclear processes including hepatocarcinogenesis as evidenced by knockout studies .

What are the validated applications for Formyl-HIST1H1C (K84) Antibody?

The Formyl-HIST1H1C (K84) Antibody has been validated for several research applications:

Other HIST1H1C antibodies (not specifically formyl-K84) have been validated for additional applications including Western Blot (1:500-1:3000), Immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg protein), Immunohistochemistry (1:100-1:600), and ChIP assays . Researchers should carefully select the appropriate antibody based on their specific experimental needs and target modification.

How does formylation at K84 affect HIST1H1C function in chromatin regulation?

Lysine formylation represents an important but less-studied histone post-translational modification compared to methylation and acetylation. Formylation of HIST1H1C at K84 likely impacts its binding affinity to DNA and interaction with chromatin remodeling complexes. Current research suggests that histone formylation may result from oxidative stress conditions, potentially serving as a cellular stress response mechanism.

The specific K84 position on HIST1H1C appears within a region that interacts with linker DNA, suggesting that formylation at this site could modulate the ability of H1.2 to compact chromatin or regulate nucleosome spacing. Researchers investigating this modification should consider combining ChIP-seq with formyl-specific antibodies to identify genomic regions where this modification predominates, and correlate with gene expression data to determine functional consequences.

What is the relationship between HIST1H1C and STAT3 signaling in cancer progression?

Research indicates a significant relationship between HIST1H1C and STAT3 signaling pathways, particularly relevant to cancer progression. Studies using HIST1H1C knockout mice have identified its role in hepatocarcinogenesis through regulation of signal transducer and activator of transcription 3 (STAT3) signaling . The H1C promoter contains multiple STAT3 binding sites (including positions -1423/-1413, -701/-691, and -162/-152) that have been confirmed through ChIP assays .

This interaction suggests that HIST1H1C expression may be regulated by STAT3 activation, creating a potential feedback loop in cancer progression. Researchers investigating cancer mechanisms should consider examining how formylation at K84 might influence this pathway, potentially altering HIST1H1C's interaction with STAT3 or affecting downstream gene expression patterns in cancer cells.

How can HIST1H1C formylation be distinguished from other post-translational modifications in multiplexed epigenetic studies?

Distinguishing histone formylation from other similar post-translational modifications presents a significant technical challenge. Researchers conducting multiplexed epigenetic studies should implement a multi-methodological approach:

• Antibody specificity validation: Perform competitive binding assays with peptides containing formyl-K84, acetyl-K84, and unmodified K84 to confirm the Formyl-HIST1H1C antibody's specificity.

• Mass spectrometry verification: Use high-resolution MS/MS to distinguish formyl (mass shift +28 Da) from acetyl modifications (mass shift +42 Da).

• Chemical labeling strategies: Employ formaldehyde-specific chemical probes that react differentially with formyl groups versus acetyl groups.

• Orthogonal techniques: Combine antibody-based detection with enzymatic assays that specifically remove formyl groups but not acetyl groups.

• Context-dependent controls: Include oxidative stress conditions known to increase formylation rates versus HDAC inhibitors that increase acetylation as experimental controls.

This comprehensive approach enables researchers to confidently identify and quantify formylation in complex epigenetic landscapes.

What are the optimal conditions for immunofluorescence assays using Formyl-HIST1H1C (K84) Antibody?

For optimal immunofluorescence results with Formyl-HIST1H1C (K84) Antibody:

Researchers should optimize these conditions based on their specific cell type and sample preparation methods. For challenging samples, consider antigen retrieval methods or alternative fixation protocols if initial results are suboptimal.

How should HIST1H1C knockout models be validated for studying histone H1.2 functions?

Proper validation of HIST1H1C knockout models is critical for accurate interpretation of experimental results. A comprehensive validation strategy includes:

• Genomic verification: Confirm the knockout at the DNA level using PCR with primers flanking the targeted region. The CRISPR/Cas9 system targeting the 5'-UTR and 3'-UTR of Hist1h1c exon 1 has been successfully used to generate knockout mice .

• Transcript analysis: Perform RT-PCR and qPCR to verify the absence of Hist1h1c mRNA.

• Protein verification: Use Western blotting with validated HIST1H1C antibodies (such as 19649-1-AP, which has been tested in multiple tissues and cell lines) to confirm the absence of H1.2 protein. The expected molecular weight is 32-33 kDa despite a calculated weight of 21 kDa.

• Compensatory mechanisms assessment: Evaluate the expression levels of other H1 variants (H1.1, H1.3, H1.4, H1.5) to identify potential compensatory upregulation.

• Phenotypic characterization: Document all observable phenotypes, including changes in chromatin compaction (using MNase assays), cell cycle progression, and tissue-specific alterations.

• Functional rescue: Perform rescue experiments by reintroducing wild-type HIST1H1C to verify that observed phenotypes are directly attributable to H1.2 deficiency.

This comprehensive validation approach ensures reliable experimental outcomes when using these models for studying histone H1.2 functions in various biological contexts.

What controls should be included when studying formylation of HIST1H1C in cellular stress responses?

When investigating HIST1H1C formylation in cellular stress responses, include these essential controls:

• Negative controls:

  • Unstressed cells to establish baseline formylation levels

  • HIST1H1C knockout or knockdown cells to confirm antibody specificity

  • Immunoprecipitation with non-specific IgG of matching species and isotype

• Positive controls:

  • Cells treated with known formylation inducers (e.g., high glucose, hypoxia)

  • Recombinant formylated HIST1H1C peptides at known concentrations

• Specificity controls:

  • Competitive inhibition with excess formylated HIST1H1C peptides

  • Parallel detection with antibodies against other HIST1H1C modifications

  • Mass spectrometry validation of formylation sites

• Procedural controls:

  • Treatment with deformylase enzymes to remove the modification

  • Time-course experiments to capture dynamic changes in formylation

  • Dose-response studies to establish threshold levels for stress-induced formylation

• Biological context controls:

  • Multiple cell types to assess tissue-specific responses

  • Inhibitors of stress-response pathways to determine mechanism specificity

  • Alternative stress inducers to test context-dependent formylation patterns

These comprehensive controls ensure robust data interpretation and minimize experimental artifacts when studying this sensitive post-translational modification.

How can researchers troubleshoot weak or non-specific signals in immunofluorescence using Formyl-HIST1H1C (K84) Antibody?

When encountering weak or non-specific signals, implement this systematic troubleshooting approach:

For optimal results, researchers should titrate the antibody concentration for each specific cell type and ensure appropriate image acquisition parameters on their microscopy systems.

How should researchers interpret discrepancies between predicted and observed molecular weights of HIST1H1C in Western blots?

Researchers commonly observe HIST1H1C running at 32-33 kDa despite its calculated molecular weight of 21 kDa . This significant discrepancy requires careful interpretation:

• Post-translational modifications: Histone proteins frequently undergo extensive PTMs (phosphorylation, acetylation, methylation, formylation) that can substantially alter their electrophoretic mobility. The formylation at K84 and other potential modifications contribute to this shift.

• Intrinsic protein properties: HIST1H1C is highly basic (high isoelectric point), which affects SDS binding and results in anomalous migration in SDS-PAGE systems. This is common for histone proteins.

• Verification approaches:

  • Use recombinant HIST1H1C protein as size reference

  • Perform mass spectrometry to confirm the actual molecular weight

  • Include HIST1H1C knockout samples as negative controls

  • Test multiple antibodies targeting different epitopes of HIST1H1C

  • Use gradient gels to improve resolution in the relevant size range

• Interpretation guidelines:

  • Report both the observed and calculated molecular weights in publications

  • Document running conditions carefully (gel percentage, buffer system)

  • Consider using specialized gel systems optimized for basic proteins

  • Validate identity through immunoprecipitation followed by mass spectrometry

This mobility shift is a consistent feature of HIST1H1C and should not be interpreted as non-specific binding if other validation criteria are met.

How can researchers quantify changes in HIST1H1C formylation levels across different experimental conditions?

For accurate quantification of HIST1H1C formylation across experimental conditions, implement this multi-faceted approach:

• Western blot quantification:

  • Use formyl-specific antibodies alongside total HIST1H1C antibodies

  • Normalize formylated signal to total HIST1H1C protein

  • Include loading controls (β-actin, GAPDH) for total protein normalization

  • Apply densitometric analysis with appropriate software (ImageJ, Image Studio)

• Immunofluorescence quantification:

  • Measure nuclear intensity of formyl-HIST1H1C staining

  • Co-stain with total HIST1H1C antibody from different species

  • Calculate the ratio of formylated/total signal per nucleus

  • Analyze >100 cells per condition for statistical robustness

• Mass spectrometry approaches:

  • Use SILAC or TMT labeling for direct comparison between conditions

  • Calculate stoichiometry of formylation at K84 relative to unmodified peptide

  • Monitor multiple HIST1H1C peptides to control for protein level changes

  • Employ parallel reaction monitoring (PRM) for highest sensitivity

• ChIP-based quantification:

  • Perform ChIP-seq with formyl-specific antibodies

  • Compare genomic distribution of formylated HIST1H1C between conditions

  • Normalize to total HIST1H1C occupancy from parallel ChIP experiments

  • Identify condition-specific changes in genomic localization

These complementary approaches provide comprehensive assessment of both global and site-specific changes in HIST1H1C formylation levels across different experimental conditions.

How can Formyl-HIST1H1C (K84) Antibody be used to investigate the role of histone formylation in cancer progression?

The Formyl-HIST1H1C (K84) Antibody offers valuable insights into cancer research through multiple methodological applications:

• Tumor tissue analysis:

  • Perform immunohistochemistry on cancer tissue microarrays to correlate formylation levels with clinical outcomes

  • Compare formylation in tumor versus adjacent normal tissue

  • Studies with HIST1H1C antibodies have successfully detected the protein in human thyroid cancer tissue and ovarian tumor tissue

• Mechanistic investigation:

  • Evaluate the relationship between HIST1H1C formylation and STAT3 signaling, as HIST1H1C has demonstrated involvement in hepatocarcinogenesis through STAT3 pathway regulation

  • Perform ChIP-seq to identify genomic regions where formylated HIST1H1C binds preferentially in cancer cells

  • Correlate with gene expression data to identify formylation-regulated oncogenes or tumor suppressors

• Cancer model systems:

  • Compare formylation levels across cancer cell lines with varying aggressiveness

  • Use HIST1H1C knockout models (generated using CRISPR/Cas9 targeting the 5′-UTR and 3′-UTR of Hist1h1c exon 1) to assess the requirement for H1.2 in tumorigenesis

  • Examine how oxidative stress conditions alter formylation patterns in cancer progression

• Therapeutic implications:

  • Test whether cancer treatments affect HIST1H1C formylation status

  • Investigate whether formylation levels predict treatment response

  • Explore the potential of targeting enzymes responsible for histone formylation

This multifaceted approach leverages the Formyl-HIST1H1C (K84) Antibody to comprehensively investigate the emerging role of histone formylation in cancer biology.

What methodologies are recommended for studying HIST1H1C in liver disease and hepatocarcinogenesis?

Based on established research showing HIST1H1C's role in hepatocarcinogenesis , the following methodologies are recommended:

• Animal models:

  • Utilize HIST1H1C knockout mice generated via CRISPR/Cas9 targeting of the 5′-UTR and 3′-UTR of Hist1h1c exon 1

  • Employ diethylnitrosamine (DEN)-induced hepatocarcinogenesis model to study tumor development

  • Monitor liver tumor formation, size, and multiplicity in wild-type versus knockout animals

  • Analyze liver function parameters (ALT, AST, bilirubin) throughout disease progression

• Histological and immunohistochemical analysis:

  • Perform H&E staining to assess liver architecture and tumor pathology

  • Use immunohistochemistry with antibodies against:

    • HIST1H1C (recommended dilution 1:100-1:600)

    • Ki-67 for proliferation assessment

    • F4/80 for macrophage infiltration

    • CD3 for T-cell infiltration

    • Ly6G for neutrophil infiltration

    • p-STAT3 Y705 to evaluate STAT3 pathway activation

• Molecular mechanisms investigation:

  • Perform ChIP assays to identify HIST1H1C binding sites in liver tissue

  • Analyze STAT3 binding to the HIST1H1C promoter at key sites (-1423/-1413, -701/-691, and -162/-152)

  • Conduct luciferase reporter assays with wild-type and mutant HIST1H1C promoters

  • Evaluate formylation status of HIST1H1C at K84 during disease progression

• Clinical translation:

  • Analyze HIST1H1C expression and formylation in human HCC tissue microarrays

  • Correlate with clinical parameters and patient outcomes

  • Investigate potential as a biomarker for disease progression or treatment response

This comprehensive methodology leverages established techniques while focusing on the specific role of HIST1H1C in liver disease pathogenesis.

How can epigenetic cross-talk between HIST1H1C formylation and other histone modifications be investigated?

Investigating epigenetic cross-talk between HIST1H1C formylation and other modifications requires sophisticated methodological approaches:

• Sequential ChIP (Re-ChIP):

  • First immunoprecipitate with Formyl-HIST1H1C (K84) Antibody

  • Elute and perform second immunoprecipitation with antibodies against other modifications

  • Sequence resulting DNA to identify genomic regions with co-occurrence

  • Alternatively, perform in reverse order to confirm bidirectional relationship

• Mass spectrometry-based approaches:

  • Perform top-down mass spectrometry on intact HIST1H1C to identify combinatorial modification patterns

  • Use middle-down approaches focusing on larger histone fragments

  • Employ electron transfer dissociation (ETD) for improved PTM identification

  • Quantify co-occurrence frequencies of formylation with other modifications

• Proximity ligation assays (PLA):

  • Use Formyl-HIST1H1C (K84) Antibody together with antibodies against other modifications

  • Visualize and quantify co-localization at the single-cell level

  • Perform under different cellular conditions to assess dynamic relationships

• Biochemical interaction studies:

  • Identify proteins that specifically recognize formylated HIST1H1C

  • Determine how these readers are affected by adjacent modifications

  • Test whether formylation affects the activity of enzymes that modify neighboring residues

• Genetic manipulation approaches:

  • Create targeted mutations that prevent specific modifications

  • Assess the impact on formylation levels and distribution

  • Use CRISPR/Cas9-based approaches to target writer enzymes for each modification

• Computational integration:

  • Develop machine learning algorithms to identify patterns in multi-modal epigenetic data

  • Predict functional consequences of specific modification combinations

  • Model the hierarchical relationships between modifications