HDAC11 Antibody

What is HDAC11 and why is it important to study?

HDAC11 (Histone Deacetylase 11), also called HD11, is the sole member of class IV histone deacetylases and functions by removing acetyl groups from histone proteins, leading to chromatin condensation and transcriptional repression. It plays crucial roles in various cellular processes including differentiation, proliferation, and apoptosis . HDAC11 is particularly important in immune cell function, where it acts as a negative regulator of IL-10 expression in antigen-presenting cells and modulates the suppressive capacity of myeloid-derived suppressor cells . It shows high expression in kidney, heart, brain, skeletal muscle, testis, and specific hematopoietic cells, making it a significant target for understanding tissue-specific gene regulation .

How do I choose the appropriate HDAC11 antibody for my experiment?

When selecting an HDAC11 antibody, consider the following methodological approach:

• Target species compatibility: Verify that the antibody recognizes HDAC11 in your species of interest. Some antibodies like the HDAC11 Antibody (C-5) detect mouse, rat, and human HDAC11 , while others may be species-specific (e.g., human-only) .

• Application suitability: Select an antibody validated for your specific application:

  • For protein detection: Western blot (WB)

  • For protein-protein interactions: Immunoprecipitation (IP)

  • For localization studies: Immunofluorescence (IF)

  • For quantitative analysis: ELISA

  • For tissue studies: Immunohistochemistry (IHC)

• Clonality consideration: Decide between monoclonal (consistent results, specific epitope) and polyclonal (higher sensitivity, multiple epitopes) based on your experimental needs .

• Epitope location: For domain-specific studies, check the epitope location. For instance, some antibodies target the N-terminus (first 300 amino acids) while others may target different regions.

• Conjugation requirements: Consider whether you need unconjugated antibodies or those conjugated with enzymes (HRP), fluorophores (FITC, PE, Alexa Fluor®), or other tags based on your detection method .

What are the typical applications for HDAC11 antibodies?

HDAC11 antibodies are versatile research tools with multiple applications:

• Western Blotting: For detecting HDAC11 protein expression levels in cell or tissue lysates. Typically observed at approximately 39-49 kDa depending on the isoform and post-translational modifications .

• Immunoprecipitation: For isolating HDAC11 protein complexes to study interaction partners and regulatory mechanisms .

• Immunofluorescence: For visualizing HDAC11 subcellular localization, which is predominantly nuclear but can vary depending on cell type and conditions .

• Chromatin Immunoprecipitation (ChIP): For identifying genomic regions where HDAC11 binds, particularly important when studying its role in regulating specific genes like IL-10 or CXCR2/CXCL2 .

• Immunohistochemistry: For examining HDAC11 expression patterns in tissue sections, especially useful in comparing normal versus pathological samples .

• Flow Cytometry: For quantifying HDAC11 expression in specific cell populations when using conjugated antibodies .

How should I validate the specificity of an HDAC11 antibody?

A methodical approach to validating HDAC11 antibody specificity includes:

• Positive controls: Use tissues/cells known to express high levels of HDAC11 (kidney, heart, brain, skeletal muscle, testis) .

• Negative controls: Include samples where HDAC11 is absent or depleted:

  • HDAC11 knockout (HDAC11KO) mouse tissues

  • Cells with HDAC11 knocked down via siRNA/shRNA

• Blocking peptide competition: Pre-incubate the antibody with the immunizing peptide to demonstrate signal specificity .

• Multiple antibody verification: Compare results using different antibodies targeting distinct HDAC11 epitopes .

• Size verification: Confirm detection at the expected molecular weight (approximately 39-49 kDa) .

• Cross-reactivity assessment: Test against related HDAC family members to ensure the antibody doesn't cross-react with other HDACs.

What are the optimal protocols for using HDAC11 antibodies in Western blotting?

For optimal Western blot results with HDAC11 antibodies:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation status

  • Sonicate briefly to shear chromatin and release nuclear proteins

• Gel electrophoresis:

  • Load 20-50 μg of total protein per lane

  • Use 10-12% SDS-PAGE gels for optimal resolution around 39-49 kDa

• Transfer conditions:

  • Semi-dry or wet transfer at 100V for 60-90 minutes

  • Use PVDF membrane for better protein retention

• Blocking:

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

  • For phospho-specific detection, use 5% BSA instead of milk

• Primary antibody incubation:

  • Dilution range: typically 1:500-1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

• Detection strategy:

  • Use HRP-conjugated secondary antibodies with enhanced chemiluminescence

  • For multiplex detection, consider fluorescently labeled secondary antibodies

• Expected results:

  • Primary band at approximately 39-49 kDa

  • Potential additional bands representing isoforms or post-translational modifications

How can I optimize immunofluorescence staining with HDAC11 antibodies?

For high-quality immunofluorescence staining with HDAC11 antibodies:

• Fixation method:

  • 4% paraformaldehyde for 15 minutes preserves structure

  • Cold methanol fixation (10 min at -20°C) may improve nuclear antigen accessibility

• Permeabilization:

  • 0.1-0.5% Triton X-100 for 10 minutes for nuclear proteins

  • Gentler 0.1% saponin can be used for cytoplasmic staining

• Antigen retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0)

  • Enzymatic retrieval with proteinase K may be necessary for some fixation methods

• Blocking:

  • 5-10% normal serum (from secondary antibody host species)

  • Add 0.1-0.3% Triton X-100 to improve penetration

• Antibody dilution and incubation:

  • Primary: 1:50-1:200 dilution, overnight at 4°C

  • Secondary: 1:500-1:1000, 1-2 hours at room temperature

• Counterstaining:

  • DAPI (1:1000) for nuclear visualization

  • Phalloidin for F-actin/cytoskeletal context

• Expected pattern:

  • Predominantly nuclear localization

  • Potential nucleolar enrichment

  • Possible cytoplasmic staining in certain cell types

What controls should I include when performing chromatin immunoprecipitation (ChIP) with HDAC11 antibodies?

For rigorous ChIP experiments with HDAC11 antibodies:

• Positive controls:

  • Input chromatin (pre-immunoprecipitation sample)

  • ChIP with antibodies against known abundant histone marks (H3K4me3)

  • PCR primers for promoters of known HDAC11 target genes (e.g., IL-10)

• Negative controls:

  • Non-specific IgG of the same species and isotype as the HDAC11 antibody

  • ChIP in HDAC11-depleted cells (siRNA knockdown or HDAC11KO)

  • PCR primers for non-target regions (gene deserts)

• Technical controls:

  • No-antibody control to check for non-specific binding to beads

  • Sonication efficiency check (200-500 bp fragments)

  • Quantitative PCR standards for accurate quantification

• Validation approaches:

  • Replicate ChIP with a different HDAC11 antibody

  • Sequential ChIP (Re-ChIP) to confirm co-localization with interacting factors

  • Comparison with published HDAC11 ChIP-seq datasets

How can I use HDAC11 antibodies to study its role in immune cell function?

To investigate HDAC11's role in immune regulation:

• Immune cell expression profiling:

  • Use flow cytometry with fluorescently-conjugated HDAC11 antibodies to quantify expression across immune cell subsets

  • Compare HDAC11 levels before and after immune activation/stimulation

• Cytokine regulation studies:

  • Immunoblot for HDAC11 in parallel with cytokine ELISAs to correlate expression with IL-10, IL-6, TNF-α production

  • ChIP with HDAC11 antibodies on cytokine promoters following different stimulation conditions

• Neutrophil function assessment:

  • Combine HDAC11 immunostaining with neutrophil migration assays

  • Measure HDAC11 levels during neutrophil extracellular trap (NET) formation

  • Correlate HDAC11 expression with changes in CXCR2/CXCL2 expression

• Myeloid cell differentiation:

  • Track HDAC11 levels during myeloid development using Western blot

  • Compare HDAC11 binding to key regulatory genes in different developmental stages

  • Use HDAC11KO models to assess differentiation potential compared to wild-type

• Co-immunoprecipitation studies:

  • Identify HDAC11 interaction partners in immune cells under different activation states

  • Compare interactomes between innate and adaptive immune cells

What approaches can I use to study HDAC11 in gene regulation and epigenetic modifications?

For investigating HDAC11's epigenetic functions:

• Global histone acetylation analysis:

  • Compare histone acetylation levels in wild-type vs. HDAC11KO cells using acetyl-specific antibodies

  • Perform mass spectrometry to identify specific lysine residues affected by HDAC11

• Integrated multi-omics approach:

  • Combine HDAC11 ChIP-seq with RNA-seq to correlate binding with transcriptional changes

  • Add ATAC-seq to assess chromatin accessibility changes around HDAC11 binding sites

  • Include histone modification ChIP-seq to characterize epigenetic landscape

• Single-cell analysis:

  • Use CUT&Tag with HDAC11 antibodies for single-cell epigenomic profiling

  • Correlate with single-cell transcriptomics to identify cell type-specific functions

• Genome-wide binding studies:

  • Perform ChIP-seq with HDAC11 antibodies under different cellular conditions

  • Analyze binding motifs to identify potential DNA-binding partners

  • Compare HDAC11 binding profiles with other HDAC family members

• Interaction with transcriptional machinery:

  • Use proximity ligation assays with HDAC11 antibodies and RNA polymerase II

  • Investigate co-occupancy with transcription factors through sequential ChIP

How can I troubleshoot weak or non-specific signals when using HDAC11 antibodies?

When encountering problems with HDAC11 antibodies:

• Weak signal troubleshooting:

  • Increase antibody concentration or incubation time

  • Try different antibody clones targeting different epitopes

  • Enhance signal detection using amplification systems (e.g., tyramide signal amplification)

  • Optimize extraction methods to ensure efficient nuclear protein isolation

  • Consider antigen retrieval methods for fixed samples

• Non-specific binding solutions:

  • Increase blocking stringency (5-10% BSA/milk, longer incubation)

  • Add 0.1-0.5% Tween-20 to wash buffers

  • Pre-absorb antibody with non-specific proteins

  • Reduce antibody concentration

  • Use more stringent washing conditions (higher salt concentration)

• Background reduction strategies:

  • Include additional blocking agents (e.g., fish gelatin, casein)

  • Extend washing steps (number and duration)

  • Filter antibody solutions before use

  • Use fresh reagents and clean equipment

• Validation approaches:

  • Test antibody on HDAC11 overexpression systems

  • Include HDAC11 knockdown/knockout controls

  • Verify with alternative detection methods

How can I utilize HDAC11 knockout models for antibody validation and functional studies?

HDAC11 knockout (HDAC11KO) models provide powerful tools for research:

• Antibody validation:

  • Use HDAC11KO tissues as negative controls to confirm antibody specificity

  • Compare staining patterns between wild-type and HDAC11KO samples to identify non-specific binding

  • Perform side-by-side Western blots of wild-type and HDAC11KO lysates

• Genotyping protocols:

  • PCR-based genotyping using specific primer sets:

    • Forward primer upstream of exon 3

    • Reverse primer 1 within exon 3

    • Reverse primer 2 downstream of exon 3

  • Expected PCR products:

    • Wild-type: 437 bp band (forward + reverse 1)

    • Homozygous knockout: 278 bp band (forward + reverse 2)

    • Heterozygous: both 437 bp and 278 bp bands

• Functional comparisons:

  • Investigate phenotypic differences between wild-type and HDAC11KO cells

  • Analyze changes in neutrophil function, migration, and phagocytosis

  • Measure alterations in cytokine production (IL-6, TNF-α, IL-10)

• Rescue experiments:

  • Reintroduce wild-type or mutant HDAC11 into knockout cells

  • Compare reconstitution efficiency using HDAC11 antibodies

  • Assess which functions can be restored with different HDAC11 variants

What are the considerations when using HDAC11 reporter systems for studying expression dynamics?

HDAC11 reporter systems enable dynamic expression monitoring:

• Available reporter models:

  • Tg-HDAC11-eGFP reporter mouse with HDAC11 promoter driving eGFP expression

  • Cell lines with fluorescent protein tags fused to HDAC11

• Genotyping reporter models:

  • PCR with primers targeting:

    • Forward primer in HDAC11 promoter 3' end

    • Reverse primer within eGFP 5' region

  • Expected product: ~400 bp band in transgenic animals, no band in wild-type

• Expression analysis approaches:

  • Flow cytometry to quantify reporter signal across cell populations

  • Live cell imaging to track HDAC11 expression dynamics

  • Tissue section fluorescence microscopy for spatial expression patterns

• Validation considerations:

  • Confirm correlation between reporter signal and endogenous HDAC11 using antibodies

  • Verify that the reporter construct doesn't alter normal HDAC11 function

  • Check for position effects that might influence expression patterns

• Experimental applications:

  • Track HDAC11 expression during cell differentiation

  • Monitor changes in expression after drug treatments

  • Isolate HDAC11-expressing cells via FACS for further analysis

How can HDAC11 antibodies be used to study its role in disease models?

HDAC11 antibodies enable investigation across various disease contexts:

• Cancer research applications:

  • Compare HDAC11 expression between normal and malignant tissues

  • Correlate HDAC11 levels with patient outcomes and treatment responses

  • Study HDAC11 in promyelocytic leukemia cell lines (e.g., HL60)

• Autoimmune disease models:

  • Examine HDAC11 expression in immune cells from patients with autoimmune conditions

  • Track changes in HDAC11 localization during inflammatory responses

  • Correlate with cytokine profiles and disease severity

• Inflammatory conditions:

  • Monitor HDAC11 levels during acute and chronic inflammation

  • Study neutrophil HDAC11 expression in inflammatory models

  • Investigate HDAC11's role in the resolution phase of inflammation

• Neurodegenerative diseases:

  • Assess HDAC11 expression in brain tissues from disease models

  • Analyze co-localization with disease-specific protein aggregates

  • Correlate with markers of neuroinflammation

• Methodological approaches:

  • Immunohistochemistry of patient tissue microarrays

  • Multiplex immunofluorescence to correlate with disease markers

  • ChIP-seq to identify disease-specific targets

What methodological considerations are important when using HDAC11 antibodies with HDAC inhibitors?

When combining HDAC11 antibodies with HDAC inhibitors:

• Timing considerations:

  • Monitor HDAC11 protein levels at multiple timepoints after inhibitor treatment

  • Assess acute versus chronic effects on expression and localization

  • Compare with transcriptional changes in HDAC11 mRNA

• Selectivity assessment:

  • Use antibodies to compare effects of pan-HDAC versus selective HDAC11 inhibitors

  • Verify target engagement through activity assays alongside expression analysis

  • Monitor effects on related HDAC family members

• Functional readouts:

  • Measure global and specific histone acetylation changes

  • Assess downstream effects on HDAC11 target genes (IL-10, CXCR2)

  • Quantify changes in neutrophil function or myeloid cell differentiation

• Experimental design:

  • Include appropriate vehicle controls

  • Use dose-response and time-course analyses

  • Consider washout experiments to assess reversibility

• Combination approaches:

  • Evaluate effects of combining HDAC11 inhibitors with other epigenetic modulators

  • Study potential synergies with conventional therapies

  • Use HDAC11 antibodies to monitor changes in protein interactions following treatment