ADNP Antibody, Biotin conjugated

What is ADNP and why is it significant in neuroscience research?

ADNP (Activity-dependent neuroprotector homeobox) is a multifunctional protein with a molecular weight of approximately 123 kDa that plays crucial roles in neuroprotection and transcriptional regulation. The protein is involved in maintaining a healthy nervous system and protecting against neurodegenerative processes . Additionally, ADNP has recently been identified as having significant functions in immune cell specialization, particularly in promoting type 2 cytokine production . This dual role in both neuronal health and immune function makes ADNP antibodies valuable tools for interdisciplinary research spanning neuroscience and immunology fields.

What are the primary applications for ADNP antibodies in research?

ADNP antibodies are employed across multiple experimental techniques, primarily:

The antibodies have demonstrated reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across species .

How does biotin conjugation enhance ADNP antibody functionality?

Biotin conjugation provides several methodological advantages for ADNP antibody applications:

• Amplified Signal Detection: The strong affinity between biotin and streptavidin/avidin (Kd ≈ 10^-15 M) allows for signal amplification in detection systems, improving sensitivity particularly in tissues with low ADNP expression.

• Multi-platform Compatibility: Biotin-conjugated ADNP antibodies can be used with various streptavidin-conjugated reporter molecules (fluorophores, enzymes, quantum dots), enabling flexibility across detection methods.

• Reduced Background: The biotin-streptavidin system often provides cleaner results with less non-specific binding compared to conventional secondary antibody approaches, particularly important when studying ADNP in complex neural tissues.

• Multiplexing Capability: Facilitates concurrent detection of ADNP with other proteins of interest in co-localization studies, which is valuable when investigating ADNP's interaction with chromatin remodeling complexes.

What are the critical factors for optimizing Western blot protocols with biotin-conjugated ADNP antibodies?

Optimizing Western blot protocols for biotin-conjugated ADNP antibodies requires attention to several parameters:

• Sample Preparation: ADNP is a high molecular weight protein (observed at 135-145 kDa) , requiring careful optimization of:

  • Gel percentage (7-8% acrylamide recommended)

  • Extended transfer times (1-2 hours at 100V or overnight at 30V)

  • Use of transfer buffers containing SDS (0.1%) to facilitate movement of large proteins

• Blocking Strategy: BSA-free formulations are recommended, as commercial BSA may contain endogenous biotin that could interfere with detection . A 5% non-fat dry milk or commercial biotin-free blockers are preferred.

• Detection System Selection:

  • For chemiluminescent detection: Streptavidin-HRP (1:5000-1:10000)

  • For fluorescent detection: Streptavidin-conjugated fluorophores (1:1000-1:2000)

• Antibody Titration: Despite manufacturer recommendations, each lab should perform titration experiments specifically for their sample types. For ADNP detection, a methodical approach testing 3-4 dilutions within the recommended range (1:500-1:8000) is advised .

• Positive Controls: Include HeLa or U-251 MG whole cell lysates as positive controls, which consistently demonstrate strong ADNP expression bands at the expected molecular weight .

How should immunofluorescence protocols be modified when using biotin-conjugated ADNP antibodies?

When conducting immunofluorescence with biotin-conjugated ADNP antibodies, consider these protocol modifications:

• Endogenous Biotin Blocking: Prior to antibody incubation, block endogenous biotin using a commercial biotin blocking kit or by sequential incubation with unconjugated avidin followed by biotin.

• Nuclear Localization Optimization: Since ADNP is predominantly localized to the nucleus , optimize permeabilization steps:

  • Use 0.1-0.3% Triton X-100 in PBS for 10-15 minutes at room temperature

  • Alternatively, employ methanol fixation (ice-cold, 10 minutes) for simultaneous fixation and permeabilization

• Signal Amplification Systems:

  • Standard: Streptavidin-conjugated fluorophore

  • Enhanced sensitivity: Tyramide signal amplification (TSA) using streptavidin-HRP followed by fluorescent tyramide substrate

  • Super-resolution applications: Use streptavidin-conjugated small fluorophores (Alexa Fluor 647 or Janelia Fluor dyes)

• Co-localization Considerations: When examining ADNP interaction with chromatin remodeling complexes like CHD4-BRG1 , select secondary detection reagents that minimize spectral overlap.

• Controls: Include peptide competition controls to validate specific nuclear staining patterns, especially important when investigating subtle changes in ADNP localization during neuronal differentiation.

What are the recommended tissue preparation methods for detecting ADNP in brain samples?

For optimal detection of ADNP in neural tissues:

• Fixation Protocol:

  • Fresh tissue: 4% paraformaldehyde, 24 hours at 4°C

  • Perfusion fixation: 4% paraformaldehyde in PBS, followed by 24-hour post-fixation

• Antigen Retrieval:

  • Primary recommendation: TE buffer pH 9.0, 95-98°C for 15-20 minutes

  • Alternative method: Citrate buffer pH 6.0, 95-98°C for 20 minutes

• Section Thickness:

  • For wide-field microscopy: 5-10 μm sections

  • For confocal microscopy: 20-40 μm sections to observe complete cellular morphology

• Signal Enhancement Strategies:

  • For low abundance detection: Use TSA amplification system

  • For aged tissue samples: Extended antigen retrieval (up to 30 minutes) may be necessary

• Recommended Controls:

  • Positive tissue control: Human hippocampus shows reliable ADNP expression

  • Negative control: Primary antibody omission

  • Absorption control: Pre-incubation with immunizing peptide

How can biotin-conjugated ADNP antibodies be utilized for ChIP-seq experiments to study chromatin remodeling?

Recent research has established ADNP's role in chromatin remodeling through interaction with the CHD4-BRG1 complex . For ChIP-seq applications:

• Cross-linking Optimization:

  • Standard formaldehyde cross-linking (1%, 10 minutes) works for strong DNA-protein interactions

  • For weaker or transient interactions, try dual cross-linking with DSG (disuccinimidyl glutarate, 2 mM) for 30 minutes followed by formaldehyde

• Chromatin Fragmentation:

  • Target fragment size: 200-500 bp

  • Sonication parameters: 25-30 cycles of 30 seconds ON/30 seconds OFF (Bioruptor)

  • Enzymatic fragmentation alternatives: Use micrococcal nuclease for more consistent fragmentation

• Immunoprecipitation Protocol:

  • Pre-clearing: Incubate chromatin with streptavidin beads to remove naturally biotinylated proteins

  • Antibody incubation: Use 5-10 μg biotin-conjugated ADNP antibody per ChIP reaction

  • Beads: Streptavidin magnetic beads provide optimal capture efficiency

• Sequential ChIP Considerations:

  • For studying ADNP co-occupancy with transcription factors (e.g., GATA3 in Th2 cells )

  • First IP: Anti-ADNP biotin-conjugated antibody

  • Elution: Mild elution with 10 mM DTT rather than harsh SDS elution

  • Second IP: Antibody against suspected co-factor

• Data Analysis Focus:

  • Motif analysis should include homeobox binding sites

  • Integration with histone modification data (particularly H3K4me3 and H3K27ac)

  • Correlation with expression data from ADNP-deficient models

What strategies can address potential data inconsistencies when comparing ADNP antibody results across different applications?

Researchers often encounter discrepancies when comparing ADNP antibody results between techniques. Here are strategies to reconcile inconsistencies:

• Molecular Weight Discrepancies:

  • Expected calculated weight: 123 kDa

  • Observed range: 135-145 kDa

  • Resolution approach: Post-translational modifications (particularly phosphorylation) can account for this discrepancy; confirm with phosphatase treatment of samples

• Antibody Epitope Accessibility Issues:

  Fixation Method	Epitope Accessibility	Recommended Applications
  Paraformaldehyde	Good for C-terminal epitopes	IF, IHC
  Methanol	Better for internal epitopes	Flow cytometry, IF
  Unfixed (native)	Best for conformational epitopes	IP, ChIP

• Validation Through Multiple Approaches:

  • Genetic validation: Compare wild-type vs ADNP-deficient samples (e.g., using Cd4CreAdnpfl/fl mouse models )

  • Peptide competition: Pre-incubation with immunizing peptide should abolish specific signal

  • Cross-antibody validation: Test with antibodies targeting different ADNP epitopes

• Protocol-Specific Considerations:

  • For IHC/IF: Antigen retrieval method dramatically affects results; compare TE buffer pH 9.0 vs citrate buffer pH 6.0

  • For IP: Buffer ionic strength affects complex stability; test multiple conditions when studying ADNP interaction with CHD4-BRG1 complex

• Reporting Standards:

  • Document antibody catalog numbers, lot numbers, and dilutions

  • Specify exact sample preparation methods

  • Include positive and negative controls in all data presentations

How can ADNP antibodies be incorporated into multiplex immunoassays to study its role in both neuronal and immune functions?

To study ADNP's dual role in neuronal protection and immune regulation , multiplex approaches are valuable:

• Multiplex Immunofluorescence Strategies:

  • Tyramide-based sequential staining: Allows for up to 6-7 targets on the same tissue section

  • Antibody stripping between rounds: Use glycine-SDS buffer (pH 2.0) or commercial antibody stripping solutions

  • Spectral unmixing: Utilize multispectral imaging systems to separate closely overlapping fluorophores

• Flow Cytometry Applications:

  • For cell type-specific ADNP detection in complex populations (brain or immune tissues)

  • Surface marker panel: CD4, CD8, B220, CD11c, GFAP (depending on target cell types)

  • Intracellular ADNP detection: After surface staining, fix/permeabilize with commercial kits optimized for nuclear proteins

  • Recommended biotin-streptavidin conjugates: APC or BV421 for minimal overlap with common surface marker fluorophores

• Cytokine-Transcription Factor Correlations:

  • For studying ADNP's role in Th2 cytokine production :

  Target	Purpose	Optimal Fluorophore Pair
  ADNP	Transcription factor	Biotin-Streptavidin-BV421
  GATA3	Th2 lineage factor	AF488
  IL-13	Effector cytokine	PE
  IL-5	Effector cytokine	PE-Cy7
  IL-4	Effector cytokine	APC

• Mass Cytometry (CyTOF) Approach:

  • For high-dimensional analysis without fluorescence spillover concerns

  • Metal-tagged antibodies against ADNP plus 30-40 additional markers

  • Data analysis using dimensionality reduction (tSNE, UMAP) and clustering algorithms

  • Correlation of ADNP expression with functional markers across neural and immune cell subsets

• Single-Cell Multi-omics Integration:

  • CITE-seq or REAP-seq for simultaneous protein (including ADNP) and transcriptome analysis

  • Correlation of ADNP protein levels with target gene expression

  • Identification of cell subpopulations with unique ADNP-associated functional states

What are the most common technical challenges when using biotin-conjugated ADNP antibodies and how can they be addressed?

Researchers frequently encounter these technical issues:

• High Background Signal:

  • Problem: Endogenous biotin in tissues/cells competing with biotin-conjugated antibody

  • Solution: Implement avidin-biotin blocking step prior to primary antibody incubation

  • Alternative approach: Use different conjugate (e.g., directly labeled fluorescent antibody)

• Weak or Absent Signal:

  • Problem: Epitope masking due to fixation or processing

  • Solution: Optimize antigen retrieval; compare TE buffer pH 9.0 with citrate buffer pH 6.0

  • Alternative: Test different antibody clones targeting different ADNP epitopes

• Non-specific Bands in Western Blot:

  • Problem: Cross-reactivity or degradation products

  • Solution: Increase blocking time/concentration; optimize antibody dilution (start with 1:1000 as recommended )

  • Validation: Include ADNP-knockout or knockdown controls

• Variable Staining Intensity Across Experiments:

  • Problem: Inconsistent antibody performance between lots

  • Solution: Prepare large aliquots of working dilution; store at -20°C with 50% glycerol

  • Quality control: Include standard positive control (HeLa cells) in each experiment

• Detection System Interference:

  Interference Source	Symptoms	Solution
  Endogenous peroxidase	High background in IHC/blots	Quench with 0.3% H₂O₂ in methanol, 30 min
  Biotin-binding proteins	Non-specific signal	Pre-block with unconjugated streptavidin
  Streptavidin binding to nuclear proteins	Nuclear artifacts	Increase blocking stringency with BSA-free blockers

How can researchers validate the specificity of their ADNP antibody results in neurological and immunological research contexts?

Rigorous validation approaches include:

• Genetic Controls:

  • Positive control: Overexpression systems (transient transfection of ADNP in HEK293 cells)

  • Negative control: CRISPR/Cas9 knockout cells or tissues from Adnp-knockout mice

  • Dose-dependent controls: siRNA or shRNA knockdown with varying efficiency

• Peptide Competition Assays:

  • Pre-incubate antibody with 5-10x molar excess of immunizing peptide

  • Include both specific peptide and irrelevant peptide controls

  • Apply to all detection methods (WB, IHC, IF, IP) for comprehensive validation

• Cross-antibody Validation:

  • Compare results using antibodies targeting different ADNP epitopes

  • Expected concordance: Similar localization and molecular weight detection

  • Investigate discrepancies through epitope mapping

• Application-specific Controls:

  • For ChIP studies: IgG control, input normalization, and known ADNP target sites

  • For functional studies: Correlation of antibody staining with functional readouts (e.g., IL-13 production in Th2 cells )

• Reproducibility Across Biological Models:

  • Test in multiple cell lines with known ADNP expression (HeLa, U-251 MG, neuronal cultures)

  • Compare tissues from different species using antibodies with confirmed cross-reactivity to human, mouse, and rat ADNP

What quality control metrics should be implemented when using biotin-conjugated ADNP antibodies for publishable research?

For rigorous and reproducible research with ADNP antibodies, implement these quality control metrics:

• Antibody Characterization Documentation:

  • Complete antibody reporting table including:

    • Clone/catalog number: e.g., 17987-1-AP , NBP2-97747 , ab244286

    • Lot number: Critical for batch-dependent variations

    • Host species and clonality: Typically rabbit polyclonal for available ADNP antibodies

    • Immunogen sequence: e.g., "Recombinant Fragment Protein within Human ADNP aa 850-1050"

    • RRID identifier: e.g., AB_2222383

• Performance Metrics:

  • Signal-to-noise ratio: Minimum 5:1 for quantitative applications

  • Reproducibility: CV% <15% between technical replicates

  • Dynamic range: Linear detection range covering at least 2 orders of magnitude

  • Sensitivity: LOD determination using titrated recombinant ADNP

• Experimental Controls Table:

  Control Type	Implementation	Expected Outcome
  Positive control	HeLa or U-251 MG lysate	135-145 kDa band
  Negative control	Primary antibody omission	No specific signal
  Knockdown/knockout	siRNA or CRISPR	Signal reduction proportional to knockdown efficiency
  Loading control	Total protein normalization	Consistent loading across samples

• Biotin Conjugation Quality Metrics:

  • Degree of labeling (DOL): Optimal range 4-8 biotin molecules per antibody

  • Post-conjugation functionality: Comparison with unconjugated antibody performance

  • Stability assessment: Performance monitoring after repeated freeze-thaw cycles

• Method-specific Validations:

  • WB: Linearity of detection with protein loading titration

  • IHC/IF: Z-stack confirmation of true nuclear localization

  • IP: Mass spectrometry confirmation of pulled-down protein identity

  • ChIP: qPCR validation of enrichment at known ADNP binding sites

How can biotin-conjugated ADNP antibodies facilitate research into ADNP syndrome and related neurodevelopmental disorders?

ADNP syndrome is a rare neurodevelopmental disorder caused by mutations in the ADNP gene. Biotin-conjugated ADNP antibodies can advance this research through:

• Patient-derived Cell Model Characterization:

  • iPSC-derived neurons from ADNP syndrome patients

  • Quantitative assessment of ADNP protein levels, localization, and co-factor interactions

  • Comparison with isogenic corrected lines to establish causality

• Mutation-specific Effects on Protein Function:

  • Detection of truncated ADNP proteins resulting from frameshift mutations

  • Analysis of mutant ADNP cellular localization

  • Co-immunoprecipitation studies to assess altered protein-protein interactions

• Animal Model Validation:

  • Confirmation of ADNP expression patterns in heterozygous ADNP mouse models

  • Correlation of protein levels with behavioral phenotypes

  • Developmental time-course studies during critical neurodevelopmental windows

• Therapeutic Development Applications:

  • Screening for compounds that stabilize mutant ADNP protein

  • Evaluation of gene therapy approaches that restore ADNP levels

  • Assessment of NAP (NAPVSIPQ) peptide effects on ADNP stability and function

• Biomarker Development:

  • Correlation of peripheral blood ADNP levels with neurological symptoms

  • Extracellular vesicle-associated ADNP as potential liquid biopsy marker

  • Longitudinal monitoring of ADNP expression during therapeutic interventions

What methodological approaches can integrate ADNP antibody-based detection with single-cell technologies to understand cell-type specific functions?

To understand cell-type specific ADNP functions:

• Single-cell Protein Analysis Platforms:

  • Imaging Mass Cytometry: Metal-tagged ADNP antibodies for tissue section analysis at subcellular resolution

  • CODEX multiplexed imaging: Up to 40-50 proteins including ADNP in spatial context

  • Microfluidic antibody capture: Quantification of ADNP from individual cells

• Multi-omics Integration Strategies:

  • CITE-seq: Surface protein + transcriptome from same cells

  • ASAP-seq: ATAC + protein including nuclear factors like ADNP

  • Spatial transcriptomics with protein detection: Visium with immunofluorescence overlay

• Neuronal Subtype Classification:

  • Brain single-cell atlas integration: Map ADNP expression across defined neuronal subtypes

  • Activity-dependent regulation: Combine ADNP detection with immediate early gene expression

  • Developmental trajectory analysis: ADNP levels across neuronal maturation states

• Immune Cell Specialization Studies:

  • Th2 cell heterogeneity: Correlation of ADNP with IL-13, IL-5, and IL-4 expression in individual cells

  • Clonal analysis: T cell receptor sequencing paired with ADNP protein levels

  • Antigen-specific responses: ADNP dynamics in tetramer-positive T cells

• Computational Analysis Approaches:

  • Trajectory inference: Pseudotime ordering of cells based on ADNP and other markers

  • Regulatory network reconstruction: ADNP as node in cell-type specific gene regulatory networks

  • Causal inference: Interventional data analysis for ADNP-dependent cellular states

How can researchers effectively study the interplay between ADNP and chromatin remodeling complexes in regulating gene expression?

Recent findings demonstrating ADNP's recruitment of the CHD4-BRG1 complex open new research directions:

• Proximity Ligation Assay (PLA) Applications:

  • Direct visualization of ADNP-CHD4 or ADNP-BRG1 interactions in situ

  • Quantification of interaction frequency across cell types and states

  • Triple-PLA to simultaneously detect ADNP-CHD4-BRG1 complex formation

• ChIP-seq and Related Technologies:

  • Sequential ChIP (ChIP-reChIP): ADNP followed by CHD4 or BRG1

  • CUT&RUN or CUT&Tag: Higher resolution mapping of ADNP binding sites

  • HiChIP: Integration of chromatin interaction data with ADNP binding

• Functional Genomics Approach:

  • CRISPR interference at ADNP binding sites: Effect on chromatin accessibility and gene expression

  • Protein domain mutations: Structure-function analysis of ADNP domains required for chromatin remodeler recruitment

  • Inducible degradation systems: Acute ADNP depletion to study immediate effects on chromatin status

• Chromatin Accessibility Analysis:

  • ATAC-seq in ADNP-deficient versus wild-type cells

  • DNase hypersensitivity with ADNP ChIP-seq overlay

  • Histone modification changes (H3K27ac, H3K4me3) following ADNP manipulation

• Live-cell Imaging Strategies:

  • FRAP (Fluorescence Recovery After Photobleaching): ADNP dynamics at chromatin

  • Single-molecule tracking: ADNP search and residence times on chromatin

  • Optogenetic recruitment: Force ADNP localization to specific genomic loci and monitor chromatin changes