HDAC3 Monoclonal Antibody

What is HDAC3 and what are its primary cellular functions?

HDAC3 (histone deacetylase 3) is a class I histone deacetylase that catalyzes the deacetylation of lysine residues on the N-terminal part of core histones (H2A, H2B, H3, and H4), as well as non-histone substrates . Unlike other class I HDACs, HDAC3 carries nuclear export and localization signals, allowing it to shuttle between the nucleus and cytoplasm .

HDAC3 functions include:

• Transcriptional regulation through histone deacetylation, which provides a tag for epigenetic repression

• Cell cycle progression regulation

• Modulation of developmental events

• Down-regulation of p53 function, affecting cell growth and apoptosis

• Participation in the circadian clock regulation

• Regulation of lipid metabolism and inflammatory responses

How do HDAC3 monoclonal antibodies differ from polyclonal antibodies in research applications?

Monoclonal HDAC3 antibodies offer several methodological advantages over polyclonal alternatives:

• Specificity: Monoclonal antibodies target a single epitope on HDAC3, reducing cross-reactivity with other HDAC family members. For example, antibody 67151-1-Ig specifically targets HDAC3 in WB, IP, and ELISA applications with confirmed reactivity in human, mouse, and rat samples .

• Reproducibility: Being derived from a single B-cell clone, monoclonal antibodies provide consistent lot-to-lot performance, essential for longitudinal studies examining HDAC3 expression or activity.

• Background reduction: The single-epitope specificity typically results in cleaner Western blots and immunostaining, particularly important when examining HDAC3 in complex tissue samples where other HDACs are expressed.

• Application versatility: Most characterized HDAC3 monoclonal antibodies are validated across multiple applications. For instance, the Y415 rabbit monoclonal antibody is suitable for IHC-P, IP, WB, ICC/IF, and Flow Cytometry .

What are the typical reactivity profiles for HDAC3 monoclonal antibodies?

HDAC3 monoclonal antibodies show various reactivity profiles that researchers should consider when selecting antibodies for specific model systems:

When working with less common model organisms, researchers should conduct preliminary validation studies, as reactivity may extend beyond the tested species based on sequence homology.

How do enzymatic versus non-enzymatic functions of HDAC3 impact experimental design when using HDAC3 antibodies?

Recent research has revealed that HDAC3 possesses both enzymatic and non-enzymatic functions, which significantly impacts experimental design and interpretation when using HDAC3 antibodies .

Methodological considerations:

• Distinguishing between functions: When designing experiments to study HDAC3, researchers should consider whether they're focusing on enzymatic activity or protein-protein interactions. Recent findings demonstrate that genetically abolishing HDAC3 enzymatic activity without affecting protein levels does not cause cardiac dysfunction on high-fat diet, while complete HDAC3 depletion does cause cardiac hypertrophy and contractile dysfunction .

• Antibody selection strategy: For enzymatic activity studies, antibodies recognizing catalytic domains (such as those near Y298) are preferable. For scaffolding/non-enzymatic functions, antibodies targeting interaction domains (such as the N-terminal region 1-122, which interacts with PP4c) may be more appropriate .

• Complementary approaches: Researchers should combine antibody-based detection with functional assays. For example, when studying HDAC3's role in transcriptional regulation, combine ChIP assays using HDAC3 antibodies with histone acetylation analysis.

• Mutation-specific considerations: When working with HDAC3 mutants, different antibody epitopes may be affected. The Y298H mutation abolishes enzyme activity while maintaining protein interactions, while the Δ33-70 deletion disrupts interaction with LAP2β without affecting NCOR1/TBL1XR1 binding .

What are the critical binding partners of HDAC3 and how do they affect antibody selection for co-immunoprecipitation studies?

HDAC3 forms functional complexes with several key binding partners that affect its activity and localization. Careful antibody selection is essential for successful co-immunoprecipitation studies:

Key binding partners and domains:

• NCoR1/SMRT (NCoR2) complex: HDAC3 is primarily found in complex with these co-repressors that regulate its enzymatic activity . The K25A mutation of HDAC3 disrupts interaction with NCOR1/TBL1XR1 .

• LAP2β (lamina-associated polypeptide 2β): Interacts with HDAC3 within residues 33-70. This interaction is independent of NCOR binding and is not affected by HDAC inhibitors SAHA or MS-275 .

• YY1 (zinc-finger transcription factor): HDAC3 binding to YY1 regulates transcription .

• PP4c (Protein Phosphatase 4 catalytic subunit): The region comprising residues 1-122 of HDAC3 is both necessary and sufficient for HDAC3-PP4c interaction .

Methodological recommendations for co-IP:

• Select antibodies with epitopes distant from known protein-interaction domains to avoid competitive binding

• For LAP2β interactions, avoid antibodies targeting residues 33-70

• For NCoR/SMRT interactions, avoid antibodies targeting regions near K25

• Validate antibody compatibility with IP buffer conditions, particularly detergent concentrations

• Consider using tagged HDAC3 constructs for challenging interactions

How does HDAC3 phosphorylation status affect antibody recognition and experimental outcomes?

HDAC3 activity is regulated by both phosphorylation and dephosphorylation, which can affect antibody epitope accessibility and experimental outcomes :

• Phosphorylation sites: Protein kinase CK2 phosphorylates HDAC3 at specific residues, which can modify protein conformation and potentially mask antibody epitopes .

• Methodological approach: When studying phosphorylated forms of HDAC3:

  • Use phospho-specific antibodies when available

  • Consider phosphatase inhibitors in lysis buffers

  • For complete analysis, compare results with and without phosphatase treatment

  • Use Phos-tag gels to separate phosphorylated from non-phosphorylated forms

• Activity correlation: Correlate antibody detection with functional assays measuring HDAC3 activity to determine the relationship between phosphorylation status and enzymatic function.

• Interaction dynamics: Phosphorylation may affect HDAC3 interactions with partner proteins like PP4c. When investigating these interactions, researchers should control for phosphorylation status using appropriate inhibitors or phosphomimetic mutants.

What are the optimal protocols for using HDAC3 monoclonal antibodies in Western blot applications?

Achieving reliable and reproducible results with HDAC3 monoclonal antibodies in Western blot applications requires specific methodological considerations:

Sample preparation:

• Use RIPA or NP-40 based lysis buffers with protease inhibitors

• Include phosphatase inhibitors to preserve phosphorylation status

• Sonicate samples briefly to shear DNA and release chromatin-bound HDAC3

• Heat samples at 95°C for 5 minutes in reducing Laemmli buffer

Western blot protocol optimization:

• Load 20-40 μg of total protein per lane for cell lysates

• Use 10-12% polyacrylamide gels for optimal resolution of HDAC3 (49 kDa)

• Transfer to PVDF membranes (preferred over nitrocellulose for HDAC3)

• Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

Antibody conditions:

• Primary antibody dilutions:

  • 67151-1-Ig: 1:1000-1:6000

  • ab32369 (Y415): Follow manufacturer recommendations (typically 1:1000)

  • MA5-15442: Follow manufacturer recommendations

• Incubate overnight at 4°C with gentle rocking

• Use TBS-T with 0.1% Tween-20 for washing steps (5 × 5 minutes)

• Secondary antibody: HRP-conjugated anti-mouse or anti-rabbit IgG at 1:5000-1:10000

Detection and validation:

• Expected molecular weight: 49 kDa

• Positive controls: HeLa cells, HSC-T6 cells, HEK-293 cells, Jurkat cells

• Consider using HDAC3 knockout/knockdown samples as negative controls

How should researchers optimize Chromatin Immunoprecipitation (ChIP) protocols when using HDAC3 monoclonal antibodies?

ChIP with HDAC3 monoclonal antibodies requires special considerations due to HDAC3's chromatin-modifying activity and complex formation:

Cross-linking optimization:

• Standard formaldehyde cross-linking (1% for 10 minutes) works for most applications

• For detection of transient HDAC3 interactions, consider dual cross-linking with DSG (disuccinimidyl glutarate) followed by formaldehyde

• Quench with 125 mM glycine for 5 minutes

Chromatin preparation:

• Sonication parameters: 30 seconds ON/30 seconds OFF for 10-15 cycles to achieve fragments of 200-500 bp

• Confirm fragmentation by agarose gel electrophoresis prior to immunoprecipitation

• Pre-clear chromatin with protein A/G beads to reduce background

Immunoprecipitation:

• Antibody amount: 3-5 μg of HDAC3 monoclonal antibody per ChIP reaction

• Include IgG control of the same isotype (e.g., Mouse IgG2b for 67151-1-Ig)

• Include positive control antibodies (e.g., anti-H3)

• Incubate overnight at 4°C with rotation

Washing and elution:

• Use stringent washing conditions to minimize background

• Perform RNase A and Proteinase K digestion steps

• Reverse cross-links overnight at 65°C

• Purify DNA using phenol-chloroform extraction or column-based methods

Controls and validation:

• Perform qPCR on known HDAC3 target genes

• Include input samples at different dilutions for quantification

• Consider sequential ChIP (Re-ChIP) to identify co-occupancy with NCoR/SMRT complex members

What are the recommended methods for studying HDAC3 enzymatic activity versus protein-protein interactions?

Different experimental approaches are required to distinguish between HDAC3's enzymatic function and its protein scaffolding roles:

For enzymatic activity assessment:

• In vitro HDAC activity assays:

  • Commercial fluorometric or colorimetric kits using synthetic acetylated substrates

  • Assay conditions: pH 8.0, presence of HDAC inhibitors as controls

  • Include recombinant HDAC3 with NCoR/SMRT deacetylase activation domain for full activity

• Cellular deacetylase activity:

  • Immunoprecipitate HDAC3 using monoclonal antibodies and measure activity of the immune complex

  • Compare wild-type HDAC3 with enzymatically inactive Y298H mutant as control

  • Use class I HDAC inhibitors (e.g., MS-275) as negative controls

For protein-protein interactions:

• Co-immunoprecipitation:

  • Use antibodies targeting different HDAC3 domains to pull down protein complexes

  • The N-terminal region (1-122) is critical for PP4c interaction

  • Residues 33-70 are essential for LAP2β binding

  • Use mild lysis conditions to preserve protein complexes

• Proximity ligation assays:

  • For in situ detection of HDAC3 interactions with partners like NCoR1, LAP2β

  • Requires validated antibodies from different species for the protein pair

• Domain mapping:

  • Use deletion mutants (e.g., Δ33-70) or point mutants (e.g., K25A) to disrupt specific interactions

  • For rescue experiments, use enzyme-dead HDAC3 mutant (Y298H) to distinguish between enzymatic and non-enzymatic functions

Why might researchers observe different molecular weights for HDAC3 in Western blot analysis?

Discrepancies in HDAC3 molecular weight detection can arise from several factors that researchers should systematically address:

Expected molecular weight: The calculated molecular weight of HDAC3 is 49 kDa , but researchers may observe bands at different sizes due to:

• Post-translational modifications:

  • Phosphorylation can cause mobility shifts, usually increasing apparent molecular weight

  • HDAC3 is regulated by phosphorylation , and multiple phosphorylation sites may exist

  • Solution: Include phosphatase treatment in parallel samples for comparison

• Alternative splicing:

  • Variant HDAC3 isoforms may exist in different tissues

  • Solution: Verify antibody epitope location relative to known splice junctions

• Proteolytic processing:

  • Sample preparation without adequate protease inhibitors may result in degradation

  • Solution: Use fresh protease inhibitor cocktail in all buffers and keep samples cold

• Antibody specificity:

  • Different monoclonal antibodies target different epitopes, potentially affecting detection

  • Solution: Validate with multiple antibodies targeting different regions of HDAC3

  • Use HDAC3 knockout/knockdown samples as negative controls

• Gel system variables:

  • Different gel percentages and buffer systems affect protein migration

  • Solution: Use protein ladder with consistent migration patterns and optimize gel percentage (10-12% recommended for HDAC3)

How can researchers address non-specific binding when using HDAC3 monoclonal antibodies?

Non-specific binding is a common challenge when working with HDAC3 antibodies due to sequence conservation among HDAC family members. Methodological approaches to minimize this issue include:

• Antibody selection and validation:

  • Choose monoclonal antibodies with demonstrated specificity (e.g., 67151-1-Ig, ab32369)

  • Verify specificity using HDAC3 knockout/knockdown controls

  • Test antibodies against recombinant HDAC1, HDAC2, and HDAC3 to confirm specificity

• Blocking optimization:

  • Test different blocking agents (BSA vs. non-fat milk)

  • Increase blocking time to 2 hours at room temperature

  • Add 0.1-0.5% Triton X-100 to blocking solution to reduce hydrophobic interactions

• Antibody dilution and incubation:

  • Titrate antibody concentration to find optimal signal-to-noise ratio

  • For 67151-1-Ig, recommended dilutions are 1:1000-1:6000 for Western blot

  • Increase washing steps (5-6 washes of 5-10 minutes each)

  • Consider lower temperature (4°C) and longer incubation times

• Sample preparation considerations:

  • Pre-clear lysates with protein A/G beads before immunoprecipitation

  • For tissue samples, perform additional centrifugation steps to remove debris

  • Consider protein extraction methods optimized for nuclear proteins

• Additional controls:

  • Include isotype controls matching the HDAC3 antibody

  • For immunofluorescence/IHC, include peptide competition controls

  • Use secondary antibody-only controls to identify non-specific binding

What factors affect HDAC3 antibody detection in different subcellular compartments?

HDAC3 shuttles between nuclear and cytoplasmic compartments , which presents unique challenges for antibody-based detection:

• Fixation and permeabilization effects:

  • Over-fixation can mask HDAC3 epitopes, particularly in protein complex regions

  • For formaldehyde fixation, limit to 10-15 minutes at room temperature

  • For methanol fixation, maintain consistent -20°C temperature and fixation time

  • Different permeabilization agents (Triton X-100, saponin) may reveal different HDAC3 pools

• Complex formation considerations:

  • HDAC3-NCoR/SMRT complexes may mask certain epitopes in the nucleus

  • HDAC3-LAP2β interaction at the nuclear envelope requires specific detection approaches

  • Solution: Use antibodies targeting different HDAC3 domains to capture all complexes

• Extraction protocol optimization:

  • For nuclear HDAC3: Use high-salt extraction buffers (>300 mM NaCl)

  • For chromatin-bound HDAC3: Include nuclease treatment in extraction

  • For cytoplasmic HDAC3: Use hypotonic lysis followed by low-speed centrifugation

• Antibody selection for localization studies:

  • Verify that the antibody epitope is accessible in different HDAC3 conformations

  • Consider using multiple antibodies targeting different regions (N-terminal vs. C-terminal)

  • For immunofluorescence, confirm specificity with siRNA/shRNA knockdown controls

How can researchers distinguish between enzymatic and non-enzymatic functions of HDAC3 in experimental systems?

Recent research has revealed the importance of HDAC3's non-enzymatic functions, particularly in cardiac tissues . Methodological approaches to distinguish these functions include:

• Genetic approaches:

  • Compare HDAC3 knockout/knockdown with enzymatically inactive HDAC3 (Y298H mutation)

  • Recent findings show that genetically abolishing HDAC3 enzymatic activity without affecting protein levels does not cause cardiac dysfunction on high-fat diet, while complete HDAC3 depletion does

  • Use the HDAC3 Δ33-70 mutant that lacks interaction with LAP2β but retains enzymatic activity

• Pharmacological approaches:

  • Compare HDAC3-selective inhibitors with pan-HDAC inhibitors

  • Analyze dose-response relationships to identify enzymatic thresholds

  • Monitor both histone acetylation and non-histone substrate acetylation

• Protein-protein interaction analysis:

  • Map interaction domains using deletion mutants and point mutations

  • Tethering experiments (e.g., tethering LAP2β to HDAC3 Δ33-70 mutant restored its ability to rescue gene expression)

  • Proximity ligation assays to visualize specific interactions in situ

• Functional readouts:

  • Gene expression analysis comparing HDAC3 depletion vs. enzymatic inhibition

  • HDAC3 depletion causes robust downregulation of lipid oxidation/bioenergetic genes and upregulation of antioxidant/anti-apoptotic genes, while enzyme activity abolishment causes much milder changes

  • Pathway-specific reporter assays to distinguish enzymatic from scaffolding effects

What are the most effective methods for studying HDAC3 in the context of inflammatory responses?

HDAC3 plays significant roles in inflammatory pathways, requiring specialized experimental approaches:

• Cell-type specific analysis:

  • HDAC3 functions differently in various immune and inflammatory cell types

  • Use cell-type specific conditional knockout models or CRISPR-mediated deletion

  • Compare effects in macrophages, T cells, endothelial cells, and tissue-resident cells

• Inflammation models:

  • Acute inflammation: LPS stimulation, TNF-α treatment, IL-1β signaling

  • Chronic inflammation: Specialized disease models (arthritis, IBD, etc.)

  • Monitor both canonical (NF-κB) and non-canonical inflammatory pathways

• HDAC3-specific molecular mechanisms in inflammation:

  • HDAC3 regulates immune modulator galectin-9 in endothelial cells

  • HDAC3 knockdown reduced IFN-γ-induced expression of galectin-9

  • HDAC3 can serve as a scaffold facilitating PI3K/IRF3 interaction

• Methodological workflow:

  • Begin with HDAC3 localization studies during inflammatory activation

  • Perform ChIP-seq to identify inflammation-specific binding sites

  • Correlate with RNA-seq to determine direct transcriptional effects

  • Validate with HDAC3 inhibitors or mutants (enzyme-dead vs. interaction-deficient)

How does HDAC3's role in circadian regulation impact experimental design and data interpretation?

HDAC3 plays a critical role in circadian regulation through both enzymatic and non-enzymatic mechanisms , which has important implications for experimental design:

• Timing considerations:

  • Control for time of day in all HDAC3 experiments, especially in liver and heart tissues

  • Document and standardize harvesting times for cells and tissues

  • Consider time-course experiments covering multiple circadian cycles (48-72 hours)

• Molecular mechanisms:

  • HDAC3 regulates both transcriptional activation and repression phases of the circadian clock in a deacetylase activity-independent manner

  • During activation phase, HDAC3 promotes accumulation of ubiquitinated BMAL1 at E-boxes

  • During repression phase, HDAC3 blocks FBXL3-mediated CRY1/2 ubiquitination and promotes CRY1-BMAL1 interaction

  • The NCOR1-HDAC3 complex regulates circadian expression of BMAL1 and genes involved in lipid metabolism in the liver

• Experimental approaches:

  • Use synchronized cell systems (serum shock, dexamethasone pulse)

  • Monitor HDAC3 chromatin occupancy across the circadian cycle using ChIP

  • Distinguish between enzymatic and scaffolding functions using enzyme-dead mutants

  • Correlate with metabolic parameters, particularly in liver and cardiac tissues

• Data interpretation:

  • Control for circadian effects when analyzing HDAC3 target gene expression

  • Consider how time of treatment affects response to HDAC inhibitors

  • Account for tissue-specific circadian patterns of HDAC3 activity and localization