HDAC4 Antibody

What is the functional significance of HDAC4 and why is it important to study?

HDAC4 belongs to the Class IIa family of histone deacetylases that plays a vital role in transcriptional regulation, cell cycle progression, and developmental events. Research indicates that HDAC4 provides neuroprotection against apoptosis in cerebellar granule neurons and cortical neurons exposed to various stress conditions . This protection appears independent of its deacetylase activity, as protective functions do not require the catalytic domain and cannot be inhibited by chemical HDAC inhibitors .

HDAC4 functions through large multiprotein complexes and is involved in:

• Muscle maturation via interaction with myocyte enhancer factors (MEF2A, MEF2C, and MEF2D)

• MTA1-mediated epigenetic regulation of ESR1 expression in breast cancer

• Deacetylation of HSPA1A and HSPA1B at 'Lys-77', affecting their binding to co-chaperone STUB1

What types of HDAC4 antibodies are commercially available and how do they differ?

Multiple HDAC4 antibody formats exist with distinct characteristics suitable for different experimental applications:

How should I select the appropriate HDAC4 antibody for my specific research application?

Selection should be based on several critical factors:

• Experimental technique: Choose antibodies validated for your specific application (WB, IP, ICC/IF, or flow cytometry)

• Species reactivity: Ensure the antibody recognizes HDAC4 in your experimental model (human, mouse, rat)

• Epitope recognition: Consider the immunogen region—antibodies targeting different domains may yield different results

• Validation status: Prioritize antibodies with published validation data and citation history

• Clonality considerations:

  • Polyclonal antibodies (e.g., ab12172) recognize multiple epitopes, potentially providing stronger signals

  • Monoclonal antibodies (e.g., ab234084) offer higher specificity for a single epitope

What are the essential controls for validating HDAC4 antibody specificity?

Proper validation requires multiple control approaches:

• Positive controls: Use tissues/cells known to express HDAC4 (brain tissue, HeLa cells, NIH/3T3 cells)

• Negative controls: Include samples with:

  • No primary antibody

  • Isotype control antibody

  • Tissues from HDAC4 knockout models when available

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity

• siRNA/shRNA knockdown: Compare staining between HDAC4-depleted and control samples

• Overexpression system: Test reactivity in HDAC4-transfected cells, as demonstrated with HDAC4 (aa 456-592)-hIgGFc-transfected HEK-293 cells

What are the optimal protocols for detecting HDAC4 by Western blotting?

Successful Western blotting for HDAC4 requires careful optimization:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylated HDAC4

  • Fractionate nuclear and cytoplasmic compartments if studying subcellular localization

• Gel selection and transfer:

  • Use 6-8% gels or gradient gels to resolve HDAC4 (predicted MW: 119 kDa)

  • Transfer to PVDF membrane at low amperage overnight for large proteins

• Antibody dilutions:

  • For ab12172: 1/500 to 1/2000 for rat brain or NIH/3T3 samples

  • For ab234084: 1/100 for HEK-293 cell lysates

• Detection method:

  • ECL-based systems work well for standard detection

  • Consider fluorescent secondary antibodies for multiplex detection with loading controls

How can I optimize immunoprecipitation protocols for HDAC4 protein complexes?

Immunoprecipitation of HDAC4 and associated proteins requires:

• Lysis conditions:

  • Use gentle lysis buffers (150-300 mM NaCl, 1% NP-40 or Triton X-100)

  • Add protease/phosphatase inhibitors and HDAC inhibitors if studying acetylation states

• Antibody amounts:

  • For ab12172: 5-10 μg per IP reaction with HeLa whole cell extract

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

• Controls to include:

  • IgG control using same species as primary antibody

  • Input sample (5-10% of starting material)

  • No cell extract negative control

• Elution and detection:

  • Gentle elution with peptide competition for native complexes

  • Standard SDS elution for standard IP-Western applications

What approach should I use for immunofluorescence detection of HDAC4?

HDAC4 shuttles between nucleus and cytoplasm, requiring careful IF protocols:

• Fixation and permeabilization:

  • 4% paraformaldehyde (10-15 minutes)

  • Permeabilize with 0.1-0.3% Triton X-100

  • Consider methanol fixation for certain epitopes

• Blocking and antibody incubation:

  • Block with 5-10% serum or BSA for at least 1 hour

  • For ab12172: Use 1/250 dilution for paraffin-embedded sections

  • For ab234084: Follow fluorescence validation protocols shown in staining images

• Counterstaining recommendations:

  • Nuclear counterstain (DAPI or DRAQ5)

  • Cytoskeletal markers (e.g., AlexaFluor-555-Phalloidin for actin)

• Imaging considerations:

  • Capture z-stacks to accurately assess nuclear vs. cytoplasmic localization

  • Use confocal microscopy for subcellular localization studies

How should I design flow cytometry experiments using HDAC4 antibodies?

Flow cytometry for HDAC4 requires careful attention to:

• Cell preparation:

  • Fixation with 2-4% paraformaldehyde

  • Permeabilization with 0.1% saponin or Triton X-100

  • Suspension cells (like Jurkat) work well for HDAC4 flow cytometry

• Antibody selection and dilution:

  • For ab234084: Use 1/200 dilution as validated in HeLa cells

  • Include isotype control matched to primary antibody

• Gating strategy:

  • Gate live cells based on forward/side scatter

  • Exclude doublets

  • Compare to negative controls to set positive gates

• Data analysis:

  • Report median fluorescence intensity

  • Consider comparing nuclear vs. cytoplasmic marker correlation

How can HDAC4 antibodies be utilized to study HDAC4's role in neuroprotection?

HDAC4 demonstrates neuroprotective properties independent of its HDAC catalytic domain . Advanced experimental approaches include:

• Primary neuron cultures:

  • Use cerebellar granule neurons with low potassium-induced apoptosis model

  • Employ cortical neurons with 6-hydroxydopamine-induced toxicity model

  • Monitor HDAC4 nuclear translocation during stress using immunofluorescence

• Analysis techniques:

  • Co-immunoprecipitation of HDAC4 with nuclear/cytoplasmic binding partners

  • ChIP-seq to identify HDAC4-associated gene promoters during neuroprotection

  • Live-cell imaging with fluorescently tagged HDAC4 to monitor translocation

• Experimental design considerations:

  • Compare wild-type HDAC4 vs. catalytically inactive mutants

  • Assess dependence on signaling pathways (Raf-MEK-ERK, PI-3 kinase-Akt)

  • Examine relationship with c-jun activation, which typically promotes neuronal death

What methods are available for studying HDAC4-selective protein degradation?

Recent research has developed HDAC4-selective protein degraders, providing tools for specific HDAC4 depletion :

• Available degrader compounds:

  • Hydroxamic acid (HA) degraders (compounds 5-7)

  • Trifluoromethyloxadiazole (TFMO) series (compounds 9-12)

  • Negative control compounds (8) for validation

• Quantification methods:

  • MSD (Meso Scale Discovery) platform using HDAC4 antibodies (ab12171, ab123513, sc-11418)

  • Western blot analysis with appropriate controls

  • Determination of DC50 (concentration for 50% degradation) and Dmax (maximum degradation)

• Selectivity assessment:

  • Western blot analysis for other HDACs (HDAC1, HDAC3, HDAC5, HDAC7, HDAC9)

  • Multiplexed analysis with HDAC4 and loading controls

  • Compounds 7 and 11 show good degradation selectivity despite having cross-reactivity in enzyme inhibition assays

How can I study the relationship between HDAC4 and cell cycle regulation?

HDAC4 inhibits CDK1 activity and cell cycle progression, with implications for neuronal survival :

• Experimental approaches:

  • CDK1 activity assays in the presence/absence of HDAC4

  • Cell cycle analysis by flow cytometry with propidium iodide staining

  • BrdU incorporation studies to measure DNA synthesis

• In vivo models:

  • HDAC4 knockout mice show elevated CDK1 activity and cerebellar abnormalities

  • Progressive loss of Purkinje neurons postnatally in posterior lobes

  • BrdU incorporation indicating inappropriate cell cycle progression

• Mechanistic investigations:

  • Co-immunoprecipitation of HDAC4 with cell cycle regulatory proteins

  • Analysis of HDAC4 nuclear localization during different cell cycle phases

  • Comparison with HDRP (HDAC-related protein) which protects via JNK inhibition

What techniques can reveal HDAC4's role in epigenetic regulation?

To study HDAC4's epigenetic functions:

• Chromatin immunoprecipitation approaches:

  • Standard ChIP to identify genomic binding sites

  • ChIP-seq for genome-wide binding profile

  • Sequential ChIP to identify co-occupancy with other factors (MEF2, etc.)

• Acetylation analysis:

  • Global histone acetylation assessment by western blot

  • Mass spectrometry to identify specific histone modifications

  • Site-specific acetylation antibodies to monitor H2A, H2B, H3, and H4 modifications

• Gene expression studies:

  • RNA-seq following HDAC4 manipulation

  • RT-qPCR for target genes in muscle development pathway

  • Analysis of ESR1 expression in breast cancer models

Why might I observe inconsistent HDAC4 antibody staining patterns between experiments?

Inconsistent staining can result from:

• Antibody-specific factors:

  • Lot-to-lot variability (especially with polyclonal antibodies)

  • Storage conditions affecting antibody stability

  • Freeze-thaw cycles reducing efficacy

• Biological variables:

  • HDAC4 shuttles between nucleus and cytoplasm based on cellular conditions

  • Phosphorylation status affects subcellular localization

  • Expression levels vary by cell type and physiological state

• Technical considerations:

  • Fixation methods significantly impact epitope accessibility

  • Antigen retrieval conditions for FFPE samples

  • Blocking reagents may interfere with certain epitopes

What might cause unexpected molecular weight bands when using HDAC4 antibodies?

HDAC4's predicted molecular weight is 119 kDa , but variants may appear:

• Higher molecular weight bands:

  • Post-translational modifications (phosphorylation, SUMOylation)

  • Protein complexes resistant to denaturation

  • Cross-reactivity with other Class IIa HDACs (HDAC5, HDAC7, HDAC9)

• Lower molecular weight bands:

  • Proteolytic cleavage products (biologically relevant)

  • Degradation during sample preparation (add protease inhibitors)

  • Alternatively spliced isoforms

  • Non-specific binding to breakdown products

• Resolution recommendations:

  • Use gradient gels (4-12%) for better separation

  • Include phosphatase treatment controls

  • Validate with multiple antibodies recognizing different epitopes

How can I address cross-reactivity concerns between HDAC4 and other HDAC family members?

Class IIa HDACs share significant homology, requiring careful specificity controls:

• Validation approaches:

  • Test antibodies on overexpression systems for each HDAC

  • Use HDAC-selective degraders (compounds 7 and 11) that demonstrate HDAC4 selectivity over other HDACs

  • Conduct knockdown experiments for each HDAC individually

• Experimental design strategies:

  • Use multiple antibodies targeting different epitopes

  • Include domain-specific antibodies when focusing on particular functions

  • Consider recombinant expression with epitope tags for unambiguous detection

• Data interpretation guidelines:

  • Always report the specific antibody clone/catalog number used

  • Acknowledge potential cross-reactivity limitations

  • Validate key findings with orthogonal techniques (e.g., mass spectrometry)

What approaches help resolve contradictory HDAC4 expression or localization data?

When faced with conflicting results:

• Methodological reconciliation:

  • Compare fixation and permeabilization protocols between studies

  • Evaluate antibody epitope locations relative to functional domains

  • Consider cell type-specific differences in post-translational modifications

• Biological explanations:

  • HDAC4 localization is dynamic and responsive to cellular signaling

  • Integration with MAPK and calcium-calmodulin signaling pathways affects localization

  • Cell cycle status influences HDAC4 function and localization

• Technical approaches:

  • Use fractionation to quantitatively assess nuclear vs. cytoplasmic distribution

  • Employ live-cell imaging to monitor dynamics

  • Conduct time-course experiments to capture translocation events

How might HDAC4-selective protein degraders advance therapeutic applications?

HDAC4-selective degraders represent a significant advance:

• Advantages over inhibitors:

  • Complete protein removal versus catalytic inhibition

  • Potential for greater selectivity despite similar binding profiles

  • Compounds 7 and 11 demonstrate selective HDAC4 degradation despite cross-reactive inhibition profiles

• Research applications:

  • Acute protein depletion to study temporal requirements

  • Tissue-specific HDAC4 degradation using targeted delivery

  • Chemical genetics approaches with engineered degrader sensitivity

• Therapeutic potential:

  • CNS applications leveraging HDAC4's role in neuronal survival

  • Muscle disorders related to HDAC4-MEF2 axis

  • Cardiac conditions involving hypertrophy pathways

What techniques will advance understanding of HDAC4's non-histone targets?

Beyond histones, HDAC4 deacetylates non-histone proteins:

• Identification methods:

  • Acetylome analysis following HDAC4 manipulation

  • Proximity labeling techniques (BioID, APEX) to identify interactors

  • In vitro deacetylation assays with candidate substrates

• Validation approaches:

  • Site-directed mutagenesis of acetylation sites

  • Generation of acetylation-mimetic mutants

  • Development of site-specific acetylation antibodies

• Known examples:

  • HDAC4 deacetylates HSPA1A and HSPA1B at 'Lys-77', affecting their binding to co-chaperone STUB1

  • Further studies needed to comprehensively map non-histone targets