β-hydroxybutyryl-HIST1H1D (K106) Antibody

What is the β-hydroxybutyryl-HIST1H1D (K106) antibody and what does it detect?

The β-hydroxybutyryl-HIST1H1D (K106) antibody is a polyclonal antibody that specifically recognizes the β-hydroxybutyryl post-translational modification at lysine 106 on the histone H1.3 protein (HIST1H1D). This antibody is designed to detect this specific epigenetic mark in human samples, allowing researchers to study this particular histone modification in the context of chromatin structure and gene regulation. Histone H1 proteins bind to linker DNA between nucleosomes, forming the macromolecular structure known as the chromatin fiber, and are necessary for the condensation of nucleosome chains into higher-order structured fibers .

What are the validated applications for β-hydroxybutyryl-HIST1H1D (K106) antibody?

The β-hydroxybutyryl-HIST1H1D (K106) antibody has been validated for several research applications including:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB)

• Immunocytochemistry (ICC)

The recommended dilutions for these applications are:

• WB: 1:100-1:1000

• ICC: 1:10-1:100

• ELISA: 1:2000-1:10000 (based on similar antibodies)

What is the significance of β-hydroxybutyrylation in histone research?

β-hydroxybutyrylation represents an important histone post-translational modification that links metabolism to gene regulation. This modification occurs when β-hydroxybutyrate (a ketone body produced during fasting or ketogenic diets) modifies specific lysine residues on histone proteins. The β-hydroxybutyryl-HIST1H1D (K106) antibody allows researchers to specifically investigate this modification at lysine 106 of histone H1.3. Understanding this modification is crucial for unraveling the mechanisms by which metabolic states influence chromatin structure and gene expression, particularly in contexts related to fasting, ketogenic diets, and certain metabolic disorders .

How should I optimize Western blot protocols when using β-hydroxybutyryl-HIST1H1D (K106) antibody?

When optimizing Western blot protocols with the β-hydroxybutyryl-HIST1H1D (K106) antibody, consider the following methodological approach:

• Sample preparation: Treat cells with sodium butyrate (30mM for approximately 4 hours) to enhance β-hydroxybutyrylation signal. This treatment has been validated to increase detection in cell lines such as HEK293, A549, and K562 .

• Antibody dilution: Begin with a 1:500 dilution for initial experiments, then adjust based on signal strength. The recommended range is 1:100-1:1000 .

• Secondary antibody: Use anti-rabbit IgG at approximately 1:50000 dilution conjugated to HRP for optimal detection .

• Expected band size: The predicted molecular weight for HIST1H1D is approximately 23 kDa.

• Blocking conditions: Use 5% non-fat dry milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature.

• Controls: Include positive controls (cell lysates treated with sodium butyrate) and negative controls (untreated lysates or competitive peptide blocking).

What cell lines and sample types have been validated for β-hydroxybutyryl-HIST1H1D (K106) antibody?

Based on research with similar β-hydroxybutyryl histone antibodies, the following cell lines and sample types have been validated:

For optimal results, sodium butyrate treatment is recommended to enhance β-hydroxybutyrylation levels and improve detection sensitivity .

How should I design ICC experiments using the β-hydroxybutyryl-HIST1H1D (K106) antibody?

For optimal ICC experiments with the β-hydroxybutyryl-HIST1H1D (K106) antibody, follow this methodology:

• Cell preparation: Grow cells on coverslips to 70-80% confluence. Consider treatment with sodium butyrate (30mM, 4h) to enhance signal.

• Fixation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.2% Triton X-100 in PBS for 10 minutes.

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

• Primary antibody: Apply the β-hydroxybutyryl-HIST1H1D (K106) antibody at a dilution of 1:50 (recommended range: 1:10-1:100) in blocking buffer and incubate overnight at 4°C .

• Secondary antibody: Use fluorophore-conjugated anti-rabbit IgG at manufacturer-recommended dilutions (typically 1:500-1:1000).

• Controls: Include a negative control (omitting primary antibody) and a competitive peptide blocking control. Consider parallel staining with other histone marks or nuclear markers.

• Imaging: Acquire images using confocal microscopy for optimal resolution of nuclear localization patterns.

How can I distinguish between β-hydroxybutyrylation and other acyl modifications in histone H1.3?

Distinguishing between β-hydroxybutyrylation and other acyl modifications requires careful analytical approaches:

• Mass spectrometry validation: Combine antibody-based detection with MS/MS analysis. β-hydroxybutyrylated peptides exhibit distinct HPLC elution profiles and fragmentation patterns compared to other acyl modifications. Synthetic peptides containing different modification isomers (such as K-bhb, K-2hb, K-2hib, and K-4hb) show distinct HPLC retention times .

• Competitive peptide assays: Use synthetic peptides with specific modifications to validate antibody specificity:

  • bhbQLATK vs 2hbQLATK

  • bhbQLATK vs acetylQLATK

  • bhbQLATK vs crotonylQLATK

• Metabolic regulation: Manipulate cellular metabolic states to differentially regulate acylation types:

  • Fasting/ketogenic conditions increase β-hydroxybutyrylation

  • Histone deacetylase inhibitors primarily affect acetylation

  • Measure changes in β-hydroxybutyryl-CoA vs acetyl-CoA cellular ratios

• Co-elution experiments: Compare elution profiles of in vivo-derived modified peptides with synthetic standards using high-resolution LC-MS/MS .

What are the recommended protocols for ChIP-seq using β-hydroxybutyryl-HIST1H1D (K106) antibody?

While standard ChIP-seq protocols serve as a starting point, the following optimizations are recommended specifically for β-hydroxybutyryl-HIST1H1D (K106) antibody:

• Crosslinking: Use 1% formaldehyde for 10 minutes at room temperature, followed by quenching with 125mM glycine.

• Chromatin preparation: Sonicate to generate fragments of 200-500bp. For linker histone studies, consider using MNase digestion followed by limited sonication.

• Immunoprecipitation conditions:

  • Use 4-5μg of β-hydroxybutyryl-HIST1H1D (K106) antibody per IP reaction

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

  • Include input, IgG, and parallel ChIP with established histone mark antibodies as controls

• Washing stringency: Increase washing stringency to reduce background:

  • Low Salt Wash: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, 150mM NaCl

  • High Salt Wash: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, 500mM NaCl

  • LiCl Wash: 0.25M LiCl, 1% NP-40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl

  • Two TE Washes

• Library preparation: Use standard ChIP-seq library preparation protocols with adapter ligation and PCR amplification.

• Data analysis: Compare β-hydroxybutyrylation patterns with other histone marks, particularly focusing on enhancer regions and gene bodies rather than just promoters.

How can metabolic manipulations affect β-hydroxybutyrylation at HIST1H1D (K106)?

Manipulating cellular metabolism can significantly alter β-hydroxybutyrylation patterns at HIST1H1D (K106), providing insights into the metabolic regulation of epigenetic mechanisms:

• Ketogenic conditions: Culturing cells in low-glucose, high-fat media or treating with β-hydroxybutyrate (1-5mM) for 24-48 hours increases β-hydroxybutyrylation at H1.3K106.

• Fasting simulation: Serum starvation for 12-24 hours elevates β-hydroxybutyrylation through increased ketone body production.

• HDAC inhibition: Treatment with HDAC inhibitors like sodium butyrate (30mM, 4h) enhances β-hydroxybutyrylation signals by preventing removal of the modification .

• Quantifiable changes: Relative changes in β-hydroxybutyrylation levels under various metabolic conditions:

• Enzyme manipulation: Overexpression or knockdown of acyltransferases or deacylases that regulate β-hydroxybutyrylation can provide mechanistic insights into the regulation of this modification.

What are common issues with β-hydroxybutyryl-HIST1H1D (K106) antibody and how can they be resolved?

Researchers commonly encounter these challenges when working with β-hydroxybutyryl-HIST1H1D (K106) antibody:

• Weak or absent signal in Western blots:

  • Solution: Pre-treat cells with sodium butyrate (30mM, 4h) to enhance β-hydroxybutyrylation

  • Decrease antibody dilution to 1:100

  • Extend primary antibody incubation to overnight at 4°C

  • Use enhanced chemiluminescence detection systems with longer exposure times

• High background in ICC experiments:

  • Solution: Increase blocking time to 2 hours

  • Use 0.3% Triton X-100 instead of 0.2% for better permeabilization

  • Add 0.1% Tween-20 to antibody dilution buffer

  • Extend washing steps to 15 minutes each, with 3-4 washes

• Cross-reactivity with other histone modifications:

  • Solution: Include competitive peptide blocking controls

  • Pre-absorb antibody with acetylated peptides to remove cross-reactive antibodies

  • Validate findings with mass spectrometry

  • Use multiple antibodies targeting different epitopes of the same modification

• Inconsistent results between experiments:

  • Solution: Standardize cell culture conditions and treatments

  • Prepare fresh buffers for each experiment

  • Store antibody in small single-use aliquots to avoid freeze-thaw cycles

  • Include internal controls in each experiment for normalization

How should β-hydroxybutyryl-HIST1H1D (K106) antibody be stored and handled to maintain optimal activity?

Proper storage and handling of the β-hydroxybutyryl-HIST1H1D (K106) antibody is crucial for maintaining its activity and specificity:

• Storage conditions:

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

  • Avoid repeated freeze-thaw cycles by preparing small single-use aliquots (5-10μL)

  • The antibody is typically provided in 50% glycerol, 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative

• Handling recommendations:

  • Thaw aliquots on ice immediately before use

  • Centrifuge briefly before opening to collect liquid at the bottom of the tube

  • Return unused portion to -20°C immediately after use

  • Do not dilute the stock antibody unless immediately using for an experiment

• Shelf life and activity monitoring:

  • Typical shelf life is 12 months when stored properly

  • Include positive controls in each experiment to monitor antibody activity over time

  • If signal diminishes, prepare fresh dilutions or use a new aliquot

• Transportation:

  • Transport on dry ice for shipments longer than 24 hours

  • Keep cold (4°C) for short transports, but avoid extended periods at room temperature

How can I validate the specificity of β-hydroxybutyryl-HIST1H1D (K106) antibody results?

Validating the specificity of β-hydroxybutyryl-HIST1H1D (K106) antibody results requires multiple complementary approaches:

• Peptide competition assays:

  • Pre-incubate antibody with excess synthetic β-hydroxybutyrylated H1.3K106 peptide (5-10μg/mL)

  • In parallel, pre-incubate with unmodified peptide or differently modified peptides

  • Specific binding should be blocked only by the exact β-hydroxybutyrylated peptide

• Mass spectrometry validation:

  • Immunoprecipitate using the antibody and analyze by LC-MS/MS

  • Compare elution profiles and fragmentation patterns with synthetic standards

  • Specific β-hydroxybutyrylated peptides should co-elute with synthetic standards and show identical fragmentation patterns

• Metabolic manipulations:

  • Signal should increase in cells treated with β-hydroxybutyrate or sodium butyrate

  • Signal should decrease in cells with knockdown of enzymes that promote β-hydroxybutyrylation

• Orthogonal antibody validation:

  • Use antibodies from different sources or that recognize different epitopes

  • Results should be consistent across different antibodies targeting the same modification

• Genetic approaches:

  • Create lysine-to-arginine mutations at the K106 site (prevents modification)

  • Signal should be abolished in the mutant, confirming specificity

How can β-hydroxybutyryl-HIST1H1D (K106) antibody be used in single-cell epigenomic studies?

Integrating β-hydroxybutyryl-HIST1H1D (K106) antibody into single-cell epigenomic workflows enables novel insights into cellular heterogeneity:

• Single-cell CUT&Tag adaptation:

  • Optimize tagmentation buffer to include 0.1% digitonin and 300mM NaCl

  • Use 500ng of β-hydroxybutyryl-HIST1H1D (K106) antibody per 100,000 cells

  • Sort cells by FACS before or after tagmentation to analyze specific populations

  • Compare β-hydroxybutyrylation patterns with cell-type-specific markers

• CITE-seq integration:

  • Conjugate β-hydroxybutyryl-HIST1H1D (K106) antibody with oligonucleotide barcodes

  • Combine with cell surface markers for multimodal analysis

  • Correlate β-hydroxybutyrylation levels with transcriptional states

• Single-cell immunofluorescence:

  • Use β-hydroxybutyryl-HIST1H1D (K106) antibody in microfluidic chambers

  • Quantify nuclear staining patterns at single-cell resolution

  • Correlate with metabolic state markers

• Spatial epigenomics:

  • Combine with spatial transcriptomics platforms

  • Map β-hydroxybutyrylation patterns within tissue architecture

  • Correlate modifications with local metabolic environments

What is the relationship between β-hydroxybutyrylation at HIST1H1D (K106) and chromatin accessibility?

The relationship between β-hydroxybutyrylation at HIST1H1D (K106) and chromatin accessibility represents an important research frontier:

• Mechanism of action: β-hydroxybutyrylation at K106 of histone H1.3 potentially weakens the interaction between the linker histone and DNA, resulting in more open chromatin configurations. This effect is distinct from acetylation, as β-hydroxybutyrylation introduces a bulkier modification that may disrupt electrostatic interactions differently.

• Experimental approaches to study this relationship:

  • Combine ChIP-seq for β-hydroxybutyryl-HIST1H1D (K106) with ATAC-seq

  • Perform salt fractionation of chromatin followed by immunoblotting

  • Use live-cell imaging with fluorescently tagged reader domains for β-hydroxybutyrylation

• Comparative analysis with other H1 modifications:

  • β-hydroxybutyrylation vs. acetylation effects on chromatin compaction

  • Interplay with H1 phosphorylation which is known to affect chromatin binding

  • Potential synergistic or antagonistic effects with core histone modifications

• Functional genomics approaches:

  • CRISPR-directed β-hydroxybutyrylation using dCas9-acyltransferase fusions

  • Targeted mutagenesis of K106 to assess local chromatin accessibility changes

  • Reader domain perturbation to identify factors recognizing this modification

How does β-hydroxybutyrylation at HIST1H1D (K106) interact with other epigenetic mechanisms in disease contexts?

β-hydroxybutyrylation at HIST1H1D (K106) interacts with other epigenetic mechanisms in complex ways that may contribute to disease pathophysiology:

• Metabolic disorders:

  • In diabetic models, altered β-hydroxybutyrate levels lead to abnormal patterns of histone β-hydroxybutyrylation

  • This affects genes involved in glucose metabolism, creating potential feedback loops

  • β-hydroxybutyryl-HIST1H1D (K106) antibody can be used to track these changes in patient-derived cells

• Neurodegenerative diseases:

  • Ketogenic diets show neuroprotective effects in some neurodegenerative disorders

  • β-hydroxybutyrylation may mediate some of these effects through epigenetic reprogramming

  • Research can examine β-hydroxybutyrylation patterns in affected vs. unaffected neurons

• Cancer metabolism:

  • Cancer cells often exhibit altered metabolic states that may affect β-hydroxybutyrylation

  • Changes in this modification could contribute to aberrant gene expression in tumors

  • Combined analysis with other epigenetic marks can reveal cancer-specific patterns

• Experimental approaches:

  • Multi-omics integration (ChIP-seq, RNA-seq, metabolomics)

  • Patient-derived samples compared with healthy controls

  • Therapeutic interventions targeting metabolic pathways with monitoring of epigenetic effects