2-hydroxyisobutyryl-HIST1H1C (K128) Antibody

What is the 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody and what does it recognize?

The 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody is a polyclonal antibody raised in rabbits that specifically recognizes the 2-hydroxyisobutyrylation modification at lysine 128 of the human histone H1.2 protein (HIST1H1C). This antibody detects a specific post-translational modification that plays roles in epigenetic regulation. The antibody specifically binds to peptide sequences surrounding the 2-hydroxyisobutyryl-Lys (128) site derived from Human Histone H1.2 .

What are the technical specifications of the 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody?

The antibody has the following specifications:

This antibody is generated using a peptide sequence surrounding the 2-hydroxyisobutyrylation site at lysine 128 of human histone H1.2 .

What is HIST1H1C and why is it significant for epigenetic research?

HIST1H1C (also known as Histone H1.2) is a linker histone variant that plays crucial roles in chromatin structure and gene regulation. It has several synonyms including H1 histone family member 2, H1.a, H12_HUMAN, H1F2, and H1s-1 . Recent research has revealed that HIST1H1C is involved in regulating autophagy and has been implicated in the development of diabetic retinopathy . As a linker histone, it influences higher-order chromatin structure and accessibility, thereby affecting transcription, DNA replication, and DNA repair processes. The post-translational modifications of HIST1H1C, including 2-hydroxyisobutyrylation at K128, represent an important epigenetic regulatory mechanism that can alter chromatin dynamics and gene expression patterns .

How should the 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody be used for immunocytochemistry (ICC)?

For immunocytochemistry applications, the following protocol is recommended:

• Sample preparation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.1% Triton X-100 for 10 minutes.

• Blocking: Block non-specific binding with 5% normal goat serum in PBS for 1 hour at room temperature.

• Primary antibody incubation: Dilute the 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody at 1:20-1:200 in blocking solution and incubate overnight at 4°C .

• Washing: Wash cells 3 times with PBS, 5 minutes each.

• Secondary antibody incubation: Apply appropriate fluorophore-conjugated secondary antibody (anti-rabbit IgG) diluted in blocking solution for 1 hour at room temperature.

• Counterstaining: Stain nuclei with DAPI and mount with anti-fade mounting medium.

• Imaging: Visualize using fluorescence or confocal microscopy.

Note that optimization of antibody concentration may be required depending on cell type and experimental conditions .

What controls should be included when using the 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody?

To ensure experimental validity and accurate interpretation of results, the following controls should be incorporated:

• Negative control: Omit primary antibody but include all other steps to assess background signal from secondary antibody and autofluorescence.

• Isotype control: Use rabbit IgG at the same concentration as the primary antibody to evaluate non-specific binding.

• Peptide competition control: Pre-incubate the antibody with excess immunizing peptide prior to immunostaining to confirm binding specificity.

• Knockdown/knockout validation: Use HIST1H1C knockdown or knockout samples to confirm antibody specificity. This can be established through siRNA transfection or CRISPR-Cas9 gene editing methods as described in research using stable HIST1H1C knockdown cell lines .

• Positive control: Include samples known to express high levels of 2-hydroxyisobutyryl-HIST1H1C (K128), such as retinal cells exposed to high glucose conditions .

How can the 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody be used to analyze autophagy?

Based on research findings that link HIST1H1C to autophagy regulation, the following methodological approach can be used:

• Transfection studies: Perform experiments involving overexpression or knockdown of HIST1H1C using vectors such as pH1.2 (for overexpression) or shRNA targeting HIST1H1C (for knockdown) .

• Autophagy marker detection: Use the 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody in conjunction with autophagy markers like LC3B-I/II conversion, SQSTM1/p62 degradation, and ATG protein expression (ATG5, ATG7, ATG12, and ATG3) .

• Fluorescence microscopy: Co-transfect cells with GFP-LC3 and assess autophagosome formation. A cell containing more than 10 cytoplasmic GFP dots is counted as an autophagic cell. Analyze at least 200 cells per treatment condition .

• Autophagy flux assessment: Treat cells with autophagy inhibitors (chloroquine at 50 μM or bafilomycin A1 at 100 nM for 12 hours) and measure SQSTM1 levels along with LC3B-I to LC3B-II conversion using western blotting .

• Correlation analysis: Correlate the levels of 2-hydroxyisobutyryl-HIST1H1C (K128) with autophagy markers to establish functional relationships.

How does 2-hydroxyisobutyrylation of HIST1H1C affect cellular pathways in diabetic retinopathy?

Research has revealed complex relationships between HIST1H1C 2-hydroxyisobutyrylation and diabetic retinopathy:

• Epigenetic regulation mechanism: Overexpression of histone HIST1H1C upregulates SIRT1 and HDAC1, which maintain the deacetylation status of H4K16. This epigenetic change leads to upregulation of ATG proteins and subsequently promotes autophagy in retinal cells .

• Inflammatory response: HIST1H1C overexpression significantly increases inflammatory marker expression, including glial fibrillary acidic protein (GFAP) in retinal Müller cells (rMC-1). It also induces transcription of inflammatory factors such as Ccl2 and Il6 .

• Cell viability impact: Elevated HIST1H1C levels significantly reduce cell viability in retinal cells. Notably, this occurs without the translocation of HIST1H1C from nucleus to cytoplasm, suggesting a mechanism distinct from previously reported apoptosis-mediating functions .

• In vivo effects: AAV-mediated HIST1H1C overexpression in the retinas leads to increased autophagy, inflammation, glial activation, and neuron loss, mirroring pathological changes identified in early diabetic retinopathy .

What experimental approaches can be used to investigate the interplay between 2-hydroxyisobutyryl-HIST1H1C (K128) and other histone modifications?

To study the relationship between 2-hydroxyisobutyryl-HIST1H1C (K128) and other histone modifications, researchers can employ these advanced approaches:

• Sequential ChIP (ChIP-reChIP): Perform chromatin immunoprecipitation first with 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody followed by antibodies against other modifications (such as acLys62, acLys96, or 2meLys45) to identify genomic regions with co-occurrence of multiple modifications .

• Mass spectrometry-based proteomics: Implement quantitative proteomics to identify and quantify combinations of post-translational modifications on HIST1H1C and determine how 2-hydroxyisobutyrylation at K128 correlates with other modifications.

• Combinatorial antibody analysis: Use the 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody alongside antibodies targeting other modifications such as HIST1H1C acLys62, acLys96, and 2meLys45 to create a comprehensive modification profile .

• Histone writer/eraser manipulation: Overexpress or knock down enzymes responsible for adding or removing 2-hydroxyisobutyryl marks, and analyze resulting changes in other histone modifications using antibody panels.

• In vitro enzymatic assays: Use recombinant enzymes to modify HIST1H1C and test whether pre-existing 2-hydroxyisobutyrylation at K128 affects the addition or removal of other modifications.

How can the 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody be utilized in genetic and pharmacological intervention studies?

For genetic and pharmacological intervention studies, consider these methodological approaches:

• siRNA-mediated knockdown: Design siRNA targeting HIST1H1C as was performed in diabetic mice retinas, where knockdown significantly attenuated diabetes-induced autophagy, inflammation, glial activation and neuron loss .

• CRISPR-Cas9 gene editing: Generate cell lines with specific mutations at the K128 site to prevent 2-hydroxyisobutyrylation and assess functional consequences using the antibody to confirm modification absence.

• Drug screening: Use the antibody to monitor changes in 2-hydroxyisobutyryl-HIST1H1C (K128) levels following treatment with candidate therapeutic compounds that may affect histone modifications.

• Stable cell line development: Establish stable HIST1H1C knockdown cell lines using shRNA as described in previous research, where cells were transfected with either blank pSuper vector or vector containing shRNA targeting HIST1H1C and selected using puromycin at 1 μg/ml .

• In vivo intervention assessment: Implement AAV-mediated delivery of HIST1H1C or HIST1H1C shRNA to animal models of disease (such as diabetic retinopathy), followed by immunohistochemical analysis using the 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody to evaluate treatment efficacy .

What are common issues encountered with the 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody and how can they be resolved?

Researchers may face several challenges when working with this antibody:

How should experimental data be interpreted when comparing different histone modifications?

When analyzing and comparing data for different histone modifications including 2-hydroxyisobutyryl-HIST1H1C (K128):

• Consider modification cross-talk: Interpret data in the context of potential interactions between different modifications. For example, research has shown that 2-hydroxyisobutyrylation of HIST1H1C affects H4K16 acetylation through SIRT1 and HDAC1 regulation .

• Account for antibody specificity: Different antibodies (e.g., those targeting acLys62, acLys96, 2meLys45, or 2-hydroxyisobutyryl-K128) may have different affinities and specificities, which can influence quantitative comparisons .

• Contextual analysis: Interpret modification patterns in the context of cellular conditions (e.g., high glucose vs. normal glucose) and functional outcomes (autophagy, inflammation, cell viability) .

• Temporal dynamics: Consider the dynamic nature of histone modifications and their potential to change rapidly in response to cellular stimuli.

• Genomic location: When performing ChIP experiments, correlate modification patterns with specific genomic regions and gene expression data to establish functional relevance.

How can conflicting results between 2-hydroxyisobutyryl-HIST1H1C (K128) levels and functional outcomes be reconciled?

When facing contradictory results:

• Validate antibody specificity: Confirm that observed signals genuinely represent 2-hydroxyisobutyryl-HIST1H1C (K128) through appropriate controls and alternative detection methods.

• Consider cell type specificity: Different cell types may exhibit different responses to HIST1H1C modification. For example, the effects observed in retinal Müller cells (rMC-1) may differ from those in other cell types .

• Examine modification density: Quantify the proportion of HIST1H1C that is 2-hydroxyisobutyrylated at K128 relative to total HIST1H1C protein levels.

• Assess experimental timing: The temporal relationship between HIST1H1C modification and downstream effects may vary, requiring time-course experiments to resolve apparent contradictions.

• Investigate compensatory mechanisms: Other epigenetic modifications or parallel pathways may compensate for changes in 2-hydroxyisobutyryl-HIST1H1C (K128), masking expected outcomes in some experimental conditions.

What emerging techniques can enhance research using the 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody?

Several cutting-edge approaches could advance research in this field:

• Single-cell epigenomics: Combine the antibody with single-cell technologies to analyze 2-hydroxyisobutyryl-HIST1H1C (K128) patterns at the individual cell level, revealing cell-to-cell heterogeneity.

• CUT&RUN or CUT&Tag: Implement these newer alternatives to traditional ChIP to achieve higher resolution and sensitivity in mapping 2-hydroxyisobutyryl-HIST1H1C (K128) genomic locations.

• CRISPR epigenome editing: Use catalytically inactive Cas9 (dCas9) fused to enzymes that add or remove 2-hydroxyisobutyryl groups at specific genomic loci, and monitor effects with the antibody.

• Live-cell imaging: Develop techniques to visualize dynamics of 2-hydroxyisobutyryl-HIST1H1C (K128) in living cells using antibody-derived detection tools.

• Spatial transcriptomics integration: Combine 2-hydroxyisobutyryl-HIST1H1C (K128) immunostaining with spatial transcriptomics to correlate modification patterns with gene expression in tissue context.

What are the therapeutic implications of targeting 2-hydroxyisobutyryl-HIST1H1C (K128) in disease states?

The potential therapeutic applications include:

• Diabetic retinopathy intervention: Research has indicated that knockdown of HIST1H1C by siRNA in the retinas of diabetic mice significantly attenuated diabetes-induced autophagy, inflammation, glial activation, and neuron loss, suggesting HIST1H1C may be a novel therapeutic target for preventing diabetic retinopathy .

• Targeted drug development: Design compounds that specifically modulate the enzymes responsible for adding or removing 2-hydroxyisobutyryl groups on HIST1H1C K128.

• Combination therapies: Develop treatment approaches that simultaneously target HIST1H1C and its downstream effectors, such as SIRT1, HDAC1, or autophagy components, for synergistic effects .

• Biomarker development: Utilize the 2-hydroxyisobutyryl-HIST1H1C (K128) Antibody to develop diagnostic tools for early detection of conditions like diabetic retinopathy by measuring modification levels in accessible samples.

• Personalized medicine applications: Stratify patients based on 2-hydroxyisobutyryl-HIST1H1C (K128) levels to identify those most likely to benefit from targeted epigenetic therapies.