Formyl-HIST1H3A (K122) Recombinant Monoclonal Antibody

What is the molecular target of Formyl-HIST1H3A (K122) antibody?

Formyl-HIST1H3A (K122) antibody specifically recognizes histone H3.1 that has been formylated at lysine residue 122. Histone H3.1 is a core component of nucleosomes, which wrap and compact DNA into chromatin, thereby regulating DNA accessibility to cellular machinery involved in transcription, replication, and repair processes . The antibody targets the peptide sequence surrounding the formyl-Lys(122) site derived from Human Histone H3, with UniProt ID P68431 . This specific post-translational modification plays a critical role in epigenetic regulation and has implications in various research areas including cancer biology and stem cell research .

How does the antibody's specificity compare between monoclonal and polyclonal versions?

Both monoclonal and polyclonal versions of Formyl-HIST1H3A (K122) antibodies are available, each with distinct characteristics:

The monoclonal antibody (such as clone 32C1) was raised against a specific peptide IMP-(Fo)K-DI formylated at K122 , providing highly reproducible results with minimal batch variation. The polyclonal version recognizes multiple epitopes around the K122 formylation site, potentially offering higher sensitivity but with greater batch-to-batch variation .

What are the optimal storage conditions for maintaining antibody activity?

To maintain optimal antibody activity, the following storage recommendations should be followed:

• Store at -20°C to -80°C for long-term storage

• Avoid repeated freeze-thaw cycles by dividing into single-use aliquots

• For short-term handling, keep on ice when not in storage

• The antibody is typically supplied in buffers containing glycerol (50%), PBS (pH 7.4), and preservatives

Some formulations contain 0.03% Proclin 300 as a preservative , while others may contain 0.035% sodium azide , which is highly toxic and should be handled with appropriate precautions.

What applications are validated for Formyl-HIST1H3A (K122) antibody?

The Formyl-HIST1H3A (K122) antibody has been validated for multiple experimental applications with different recommended protocols:

While the antibody has been validated for the above applications, researchers should perform their own validation with appropriate controls when using the antibody in new experimental systems or conditions .

How should samples be prepared for optimal detection of Formyl-HIST1H3A (K122)?

For optimal detection of formylated HIST1H3A at K122, sample preparation is critical:

• For cellular/tissue extracts (Western blot):

  • Use acid extraction methods (such as HeLa acid extract) to efficiently isolate histones

  • Include protease inhibitors and deacetylase inhibitors to prevent modification loss

  • Add formylation stabilizing agents if available to preserve the modification

  • Maintain cold temperature throughout extraction to minimize enzymatic activity

• For fixed samples (IF/IHC):

  • Optimal fixation with 4% paraformaldehyde preserves epitope accessibility

  • Permeabilization conditions should be optimized (typically 0.1-0.5% Triton X-100)

  • Antigen retrieval methods may be necessary for tissue sections (citrate or EDTA buffer)

  • Blocking with appropriate agents (BSA, normal serum) to reduce background

• For all applications:

  • Include positive controls (such as HeLa cells, which express detectable levels of the target protein)

  • Consider including a formylation-inducing treatment as a positive control

  • Include technical replicates to ensure reproducibility of results

What controls should be included when using this antibody?

• Positive controls:

  • HeLa acid extract can serve as a positive control

  • Samples treated with formylation-inducing conditions

  • Recombinant HIST1H3A with formylated K122 (if available)

• Negative controls:

  • Primary antibody omission

  • Isotype control (non-specific rabbit IgG)

  • Samples treated with deformylase enzymes

  • Peptide competition assay using the immunizing peptide

• Specificity controls:

  • Comparison with antibodies recognizing total H3

  • Use of genetic knockouts/knockdowns where feasible

  • Peptide competition with formylated versus non-formylated peptides

Why might I observe heterogeneous staining patterns in immunofluorescence experiments?

Heterogeneous staining patterns in immunofluorescence using Formyl-HIST1H3A (K122) antibody may occur for several research-relevant reasons:

• Cell cycle-dependent formylation: Histone modifications can vary throughout the cell cycle, particularly during DNA replication and mitosis. Cells in different cycle phases may show differential formylation patterns .

• Transcriptional activity variation: Since histone modifications regulate gene expression, heterogeneity may reflect differential transcriptional states among cells.

• Newly incorporated versus existing histones: The antibody may preferentially recognize newly incorporated formylated H3.1 versus older histone proteins . This is particularly relevant as one researcher specifically asked whether the antibody recognizes only newly incorporated H3.1 .

• Technical factors:

  • Incomplete permeabilization

  • Inconsistent fixation

  • Variable antibody penetration

  • Epitope masking due to chromatin compaction differences

To address this heterogeneity, researchers should:

• Synchronize cells if cell cycle effects are suspected

• Compare staining patterns with total H3 antibodies

• Optimize fixation and permeabilization protocols

• Consider chromatin decompaction methods if accessibility is an issue

How can signal-to-noise ratio be optimized in Western blots with this antibody?

To optimize signal-to-noise ratio in Western blots using Formyl-HIST1H3A (K122) antibody:

• Sample preparation optimization:

  • Use acid extraction methods specifically designed for histones

  • Include appropriate modification-preserving inhibitors

  • Consider enrichment of histone fractions before loading

• Blocking optimization:

  • Test different blocking agents (5% non-fat milk, 3-5% BSA)

  • Optimize blocking time (typically 1-2 hours at room temperature)

  • Consider specialized blocking reagents for phospho-specific antibodies

• Antibody incubation parameters:

  • Test different dilutions (starting with manufacturer recommendations)

  • Optimize incubation time and temperature (4°C overnight versus room temperature)

  • Add 0.05-0.1% Tween-20 to reduce background

• Washing optimization:

  • Increase number and duration of washes (typically 3-5 washes of 5-10 minutes each)

  • Use appropriate wash buffer (TBST or PBST with 0.05-0.1% Tween-20)

• Detection method considerations:

  • Compare ECL substrates of different sensitivities

  • Consider fluorescent secondary antibodies for greater linear range

  • Optimize exposure time to prevent overexposure

What is the significance of lysine 122 formylation in histone H3.1 for epigenetic regulation?

Lysine 122 formylation (K122fo) in histone H3.1 represents an important but less-studied post-translational modification with significant implications for epigenetic regulation:

• Structural implications: K122 is located in the globular domain of H3, at the interface between DNA and the histone octamer. Formylation at this site likely alters DNA-histone interactions, potentially affecting nucleosome stability and chromatin accessibility .

• Functional consequences:

  • Altered transcriptional regulation due to changed DNA-histone affinity

  • Potential modulation of other histone modifications in the vicinity

  • Possible recruitment of specific reader proteins that recognize formylated lysine

• Interplay with other modifications: K122 formylation may affect or be affected by other nearby modifications, creating a complex regulatory network within the histone code. This includes potential crosstalk with acetylation and methylation marks.

• Biological contexts: Formylation at K122 may play roles in:

  • Cancer progression and chemoresistance pathways

  • Stem cell dynamics and differentiation processes

  • Cellular stress responses and metabolic adaptation

Further research is needed to fully elucidate the writers (enzymes that add formyl groups), erasers (enzymes that remove them), and readers (proteins that recognize formylated lysines) in this pathway.

How does Formyl-HIST1H3A (K122) relate to cancer research applications?

Formyl-HIST1H3A (K122) has emerging significance in cancer research with several key applications:

• Biomarker potential: Altered formylation patterns may serve as cancer biomarkers. The antibody enables detection of these alterations in patient samples using IHC or other diagnostic techniques .

• Epigenetic dysregulation: Cancer cells often display epigenetic abnormalities. Studying K122 formylation may reveal specific mechanisms of gene expression dysregulation in tumor development .

• Research applications in oncology:

  • Comparing formylation levels between normal and tumor tissues

  • Correlating formylation patterns with clinical outcomes

  • Studying changes in formylation during treatment response

  • Investigating drug resistance mechanisms related to histone modifications

• Therapeutic implications: Understanding the role of K122 formylation may reveal:

  • Novel druggable targets in the formylation/deformylation pathways

  • Potential for combination therapies targeting formylation alongside other treatments

  • Predictive biomarkers for treatment response to epigenetic therapies

Current research indicates interest in breast cancer applications specifically, as noted in the antibody product specifications , suggesting particular relevance in this cancer type.

How can this antibody be used in multi-omics experimental approaches?

Integration of Formyl-HIST1H3A (K122) antibody into multi-omics research frameworks enables comprehensive understanding of the biological significance of this modification:

• Epigenomics approaches:

  • ChIP-seq to map genomic distribution of K122 formylation

  • CUT&RUN or CUT&Tag for higher resolution localization

  • Integration with other histone modification maps to understand the histone code

• Proteomics integration:

  • Immunoprecipitation followed by mass spectrometry (IP-MS) to identify proteins interacting with formylated H3K122

  • Proximity labeling approaches to capture the molecular neighborhood of formylated histones

  • Analysis of co-occurring modifications using specialized MS approaches

• Transcriptomics correlation:

  • Integration of formylation ChIP-seq data with RNA-seq to correlate with gene expression patterns

  • Single-cell approaches to understand heterogeneity in formylation and expression

• Functional genomics:

  • CRISPR screens targeting enzymes involved in formylation/deformylation

  • Perturbation studies altering metabolic pathways that influence formylation levels

• Suggested experimental workflow:

  Phase	Approach	Outcome
  Discovery	ChIP-seq with Formyl-HIST1H3A (K122) antibody	Genome-wide K122fo distribution
  Integration	Correlate with RNA-seq, other histone marks	Functional associations
  Validation	Site-directed mutagenesis, enzyme perturbation	Causal relationships
  Mechanism	IP-MS, reader domain screening	Molecular partners
  Function	Phenotypic assays after perturbation	Biological significance

This integrated approach would provide a comprehensive understanding of the biological role of H3K122 formylation beyond what single-technique approaches could achieve.

How are Formyl-HIST1H3A (K122) recombinant monoclonal antibodies produced?

The production of Formyl-HIST1H3A (K122) recombinant monoclonal antibodies involves a sophisticated multi-step process:

• Immunization and gene extraction: Rabbits are immunized with a synthetic peptide containing formylated K122 derived from human HIST1H3A. Genes encoding antibodies specific to this modification are then extracted from the rabbit immune cells .

• Recombinant expression: The extracted antibody genes are integrated into specialized expression vectors, which are then introduced into host suspension cells .

• Culture and production: The modified host cells are cultured under optimized conditions to stimulate expression and secretion of the antibody .

• Purification: Affinity chromatography techniques are employed to isolate the antibody from the cell culture supernatant . This typically involves protein A/G affinity purification followed by additional purification steps if needed.

• Quality control: The purified antibody undergoes comprehensive validation through multiple assays including ELISA, Western blotting, immunohistochemistry, and immunofluorescence to confirm specificity and functionality .

This recombinant approach offers advantages over traditional hybridoma-based monoclonal antibody production, including better reproducibility, reduced batch-to-batch variation, and elimination of hybridoma instability issues.

What are the specific buffer compositions and formulations for this antibody?

Understanding the buffer composition is essential for optimal antibody handling and application compatibility:

When designing experiments, researchers should consider buffer components that might interfere with particular applications. For example:

• Sodium azide can inhibit peroxidase activity in HRP-based detection systems

• High glycerol content may affect loading in some applications

• Salt concentration may need adjustment for certain enzymatic reactions