The Formyl-HIST1H1C (K84) Antibody is a polyclonal rabbit-derived antibody specifically targeting the formylated lysine residue at position 84 (K84) of histone H1.2, encoded by HIST1H1C. This post-translational modification (PTM) is critical for studying chromatin dynamics, epigenetic regulation, and cellular processes involving linker histones. The antibody is validated for applications such as immunofluorescence (IF) and enzyme-linked immunosorbent assay (ELISA), with a focus on detecting formylation at this site in human cells .
The antibody enables visualization of formylated H1C in nuclear compartments. For example:
Protocol: Fixed cells (e.g., HeLa) are permeabilized, blocked, and incubated with the primary antibody overnight at 4°C. Detection uses Alexa Fluor 488-conjugated secondary antibodies .
Expected Localization: Nuclear staining, consistent with histone H1's role in chromatin structure .
While not directly studied for K84 formylation, anti-histone H1 antibodies broadly inhibit dendritic cell (DC) maturation by blocking signaling pathways (e.g., p38 MAPK and NF-κB) . This suggests that formylation at K84 could influence H1's extracellular signaling roles, such as modulating immune cell activity.
Mechanistic Studies:
Therapeutic Implications:
Cross-Species Reactivity:
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.
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 .
The Formyl-HIST1H1C (K84) Antibody has been validated for several research applications:
Application | Validated | Recommended Dilution | Notes |
---|---|---|---|
ELISA | Yes | According to protocol | For quantitative detection of formylated HIST1H1C |
Immunofluorescence (IF) | Yes | 1:50-200 | Successfully used in Hela cells |
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The Formyl-HIST1H1C (K84) Antibody offers valuable insights into cancer research through multiple methodological applications:
Tumor tissue analysis:
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.
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:
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.
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