ANKS1B Antibody

What is ANKS1B and why is it important to neurodevelopmental research?

ANKS1B encodes AIDA-1 (Amyloid-beta protein intracellular domain-associated protein 1), a protein highly enriched at neuronal synapses that regulates synaptic plasticity. It is critically important in neurodevelopmental research because heterozygous deletions in the ANKS1B gene cause ANKS1B Neurodevelopmental Syndrome (ANDS), characterized by autism spectrum disorder (ASD), attention deficit/hyperactivity disorder (ADHD), and speech and motor deficits. More than 60% of ANDS patients exhibit ASD, making ANKS1B a significant gene for understanding the molecular basis of autism and related disorders . AIDA-1 is localized at postsynaptic densities (PSDs) where it binds to N-methyl-D-aspartate receptors (NMDARs) and the synaptic scaffolding protein PSD95, suggesting its importance in synaptic function and plasticity .

What domains and structures characterize the ANKS1B protein that antibodies might target?

The ANKS1B protein contains specific structural domains that antibodies can target, including one phosphotyrosine-binding (PTB) domain and two sterile alpha motif (SAM) domains . The protein functions as a scaffold at postsynaptic densities and plays a critical role in long-term potentiation (LTP), a basic mechanism underlying learning and memory . Commercial antibodies often target specific regions, such as amino acids 107-268 or 851-1000 of the human ANKS1B protein . Understanding these domains is essential when selecting antibodies for specific experimental applications, as different epitopes may be more or less accessible depending on the protein's conformation in various experimental contexts.

Beyond neuronal functions, what other biological roles of ANKS1B might researchers investigate using antibodies?

While ANKS1B is well-known for its neuronal functions, research has revealed unexpected roles in other cellular contexts. ANKS1B interacts with the cerebral cavernous malformation protein KRIT1 (CCM1) and affects endothelial cell barrier functions . Studies have shown that silencing ANKS1B expression disrupts endothelial barrier functions, leading to increased permeability, while forced ANKS1B expression reduces permeability . This function appears independent of Rho kinase activity and KRIT1 presence, suggesting ANKS1B controls endothelial permeability through a distinct mechanism from KRIT1 . Additionally, recent research has identified an unexpected role for ANKS1B in oligodendroglial development, with Anks1b-deficient mouse models displaying deficits in oligodendrocyte maturation and myelination . Researchers can use ANKS1B antibodies to investigate these diverse functions across different cell types and tissues.

What are the optimal experimental conditions for using ANKS1B antibodies in immunohistochemistry of brain tissue?

For optimal immunohistochemistry (IHC) results with ANKS1B antibodies in brain tissue, researchers should consider several critical factors. First, proper fixation is essential—typically 4% paraformaldehyde works well for preserving ANKS1B epitopes. For paraffin-embedded sections, antigen retrieval is crucial; a citrate buffer (pH 6.0) with heat-induced epitope retrieval often yields good results . Antibody dilutions typically range from 1:200-1:400 for IHC-P applications , but this should be optimized for each specific antibody and tissue. When designing controls, include both positive controls (tissues known to express high levels of ANKS1B, such as hippocampus and cortex) and negative controls (either tissues with low expression or primary antibody omission). Since ANKS1B is enriched at postsynaptic densities, co-staining with neuronal markers or synaptic proteins (like PSD95) can provide valuable context for localization studies . For fluorescent detection, minimize autofluorescence by using Sudan Black B treatment, particularly in aged brain tissue.

How should researchers optimize Western blot protocols for detecting different ANKS1B isoforms?

Optimizing Western blot protocols for ANKS1B requires attention to several technical details due to the multiple isoforms and potential post-translational modifications. Sample preparation is critical—use RIPA buffer with protease and phosphatase inhibitors for brain tissue or neuronal cultures. For protein separation, 7-10% polyacrylamide gels typically work well, although gradient gels (4-15%) may better resolve the various ANKS1B isoforms. Transfer conditions should be optimized for these larger proteins—use lower current (250-300 mA) for longer duration (90-120 minutes) or consider wet transfer overnight at 4°C for complete transfer.

For antibody dilutions, start with 1:300-1:1000 for primary antibodies and optimize as needed . Include appropriate positive controls (brain lysate) and size markers to identify specific isoforms. When analyzing results, be aware that ANKS1B has multiple splice variants that can appear at different molecular weights. The primary isoforms typically run at approximately 120-150 kDa, but post-translational modifications may affect migration patterns. If non-specific bands appear, optimize blocking conditions (5% non-fat dry milk or BSA) and consider using more stringent washing steps with higher salt TBST.

What are the best approaches for co-immunoprecipitation studies involving ANKS1B and its interaction partners?

For effective co-immunoprecipitation (co-IP) of ANKS1B and its interaction partners, researchers should consider several methodological approaches. Begin with an appropriate lysis buffer—for ANKS1B interactions, a non-denaturing buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% NP-40 or Triton X-100, with protease and phosphatase inhibitors) preserves protein-protein interactions. Pre-clear lysates with protein A/G beads to reduce non-specific binding.

For the IP step, both direct (antibody pre-bound to beads) and indirect (antibody added to lysate followed by beads) approaches work, though direct coupling may reduce background from IgG bands. When studying ANKS1B interactions with known partners like KRIT1, which binds to the PTB domain of ANKS1B , ensure the antibody's epitope doesn't overlap with interaction domains. For elution, gentle methods like competitive elution with the immunizing peptide may better preserve weak or transient interactions.

Always include appropriate controls: IgG control, input sample (5-10% of starting material), and when possible, samples with confirmed interaction (positive control) or known non-interactors (negative control). For ANKS1B interactions with amyloid-beta protein or the tyrosine kinase receptor EphA8 , consider crosslinking approaches if interactions are transient or weak.

How can single-cell analysis techniques be combined with ANKS1B antibodies to study heterogeneity in neurodevelopmental disorders?

Single-cell analysis combined with ANKS1B antibodies offers powerful approaches to understanding cellular heterogeneity in neurodevelopmental disorders. For single-cell immunofluorescence, researchers can employ CLARITY or iDISCO+ tissue clearing methods followed by immunolabeling with ANKS1B antibodies, allowing three-dimensional visualization of ANKS1B distribution in intact neural circuits. This approach is particularly valuable for studying regional differences in ANKS1B expression in conditions like ANDS .

For quantitative analysis, CyTOF (mass cytometry) can be combined with ANKS1B antibodies conjugated to rare earth metals, enabling simultaneous measurement of multiple proteins at single-cell resolution. This approach can reveal how ANKS1B alterations affect different cell populations in heterogeneous tissues like brain.

Single-cell RNA-seq paired with protein analysis (CITE-seq) using ANKS1B antibodies can correlate transcriptomic profiles with protein expression. This combined approach is particularly valuable for investigating the relationship between ANKS1B genetic variants and protein expression in patient-derived neurons or glial cells. When analyzing data from these experiments, computational approaches like trajectory analysis can reveal developmental abnormalities in ANKS1B-deficient cells, and clustering algorithms can identify distinct cellular populations affected by ANKS1B dysfunction.

What strategies can address cross-reactivity issues when using ANKS1B antibodies in tissues expressing both ANKS1B and the related ANKS1A protein?

Addressing cross-reactivity issues between ANKS1B and ANKS1A requires several strategic approaches due to their similar domain structures . First, antibody selection is critical—choose antibodies raised against regions with minimal sequence homology between ANKS1B and ANKS1A. The most specific antibodies target unique regions outside the conserved ankyrin repeats, PTB, and SAM domains. Commercial antibodies should be validated using specific controls, including tissues or cells with confirmed ANKS1B knockdown/knockout and ANKS1A knockdown/knockout.

For experimental validation, perform parallel Western blots with recombinant ANKS1B and ANKS1A proteins to assess cross-reactivity directly. Competition assays with blocking peptides specific to either protein can confirm antibody specificity. In tissues expressing both proteins, consider using genetic approaches like siRNA knockdown of either ANKS1A or ANKS1B to confirm antibody specificity . When cross-reactivity cannot be eliminated, employ double-labeling approaches with antibodies known to distinguish between the two proteins, or use in situ hybridization for ANKS1B mRNA to complement protein detection.

For data interpretation, carefully document antibody validation steps and acknowledge potential cross-reactivity limitations. When studying tissues with high expression of both proteins, consider complementary approaches like mass spectrometry-based proteomics to independently verify protein identity.

How can ANKS1B antibodies be utilized in studying the role of oligodendrocyte dysfunction in ANKS1B neurodevelopmental syndrome (ANDS)?

Recent research has revealed an unexpected role for oligodendroglial deficits in ANDS pathophysiology . To study this aspect, researchers can employ several specialized approaches using ANKS1B antibodies. For tissue-level analysis, perform double immunofluorescence labeling with ANKS1B antibodies and oligodendrocyte markers (Olig2, MBP, PLP) in brain sections from Anks1b-deficient mouse models or human ANDS patient samples. This can reveal changes in oligodendrocyte numbers and myelination patterns, as previous studies have shown decreased Olig2-positive oligodendrocytes in the corpus callosum of Anks1b haploinsufficient mice .

For cellular-level investigations, isolate primary oligodendrocyte precursor cells (OPCs) from Anks1b-deficient mice and use ANKS1B antibodies to track protein levels during differentiation. Co-culture experiments with neurons can assess whether ANKS1B dysfunction in oligodendrocytes affects myelination directly or whether neuronal ANKS1B indirectly influences oligodendrocyte maturation.

To investigate molecular mechanisms, perform co-immunoprecipitation with ANKS1B antibodies in oligodendrocyte lysates to identify binding partners specific to these cells, focusing on Rac1 pathway components given the observed Rac1 dysfunction in Anks1b-deficient models . Chromatin immunoprecipitation sequencing (ChIP-seq) using ANKS1B antibodies can identify potential transcriptional roles of ANKS1B in oligodendrocyte maturation. For therapeutic development, test whether restoring ANKS1B expression specifically in oligodendrocytes rescues myelination defects in Anks1b-deficient models, which would suggest a cell-autonomous role in these cells.

How should researchers interpret discrepancies in ANKS1B expression patterns detected using different antibodies?

When confronted with discrepancies in ANKS1B expression patterns detected by different antibodies, researchers should follow a systematic analytical approach. First, evaluate each antibody's target epitope—antibodies recognizing different domains may yield different results if epitope accessibility varies across tissues or experimental conditions. For instance, antibodies targeting amino acids 107-268 may show different patterns than those targeting the 851-1000 region .

Compare detection methods—discrepancies might arise from methodological differences rather than true biological variance. Western blots detect denatured proteins, while immunohistochemistry reveals native conformations in a cellular context. Consider post-translational modifications—some antibodies may preferentially detect phosphorylated, glycosylated, or cleaved forms of ANKS1B.

Verify results using orthogonal techniques such as RNA in situ hybridization to correlate protein with mRNA expression, or mass spectrometry to confirm protein presence and modifications. When analyzing splicing variants, consult existing databases for known ANKS1B isoforms expressed in your tissue of interest, as some antibodies may not detect all splice variants.

Document all methodological details, including fixation methods, antigen retrieval protocols, antibody dilutions, and detection systems. Finally, when publishing, transparently report discrepancies and provide detailed antibody information (catalog number, lot, validation data) to facilitate research reproducibility.

What methodological approaches can distinguish between ANKS1B dysfunction in neurons versus oligodendrocytes in mouse models of ANDS?

Distinguishing ANKS1B dysfunction between neurons and oligodendrocytes requires specialized methodological approaches. Cell-type specific conditional knockout models provide the most definitive approach—compare Anks1b knockout in neurons (using Camk2a-Cre or Nex-Cre) versus oligodendrocytes (using Pdgfrα-Cre or Sox10-Cre) to isolate cell-autonomous effects . For protein localization, perform high-resolution confocal microscopy with triple immunofluorescence using ANKS1B antibodies alongside neuron-specific (NeuN, MAP2) and oligodendrocyte-specific (CC1, Olig2) markers.

To assess functional consequences in each cell type, measure electrophysiological parameters in neurons and conduct electron microscopy to evaluate myelin ultrastructure. Cell-type specific isolation techniques like fluorescence-activated cell sorting (FACS) or immunopanning followed by biochemical analysis with ANKS1B antibodies can reveal cell-type specific binding partners and signaling pathways.

For in vivo imaging, viral-mediated expression of fluorescently-tagged ANKS1B under neuron-specific or oligodendrocyte-specific promoters can track dynamic protein localization. Importantly, researchers should compare phenotypes between global Anks1b-deficient models and cell-type specific knockouts—if oligodendrocyte-specific knockout recapitulates white matter abnormalities seen in global knockout models, this would suggest a primary role for ANKS1B in oligodendrocytes rather than a secondary effect of neuronal dysfunction .

How can researchers quantitatively compare ANKS1B protein levels across different brain regions in neurodevelopmental disorder models?

Quantitative comparison of ANKS1B protein levels across brain regions requires rigorous methodological approaches to ensure accuracy and reproducibility. For Western blot analysis, use microdissection techniques to isolate specific brain regions with anatomical precision before protein extraction. Always include internal loading controls (β-actin, GAPDH) and consider using tissue-specific housekeeping proteins, as expression of common housekeeping genes can vary across brain regions.

For spatial resolution with quantification, employ quantitative immunofluorescence with ANKS1B antibodies, using identical acquisition parameters across all samples. Include fluorescent standards in each experiment to create standard curves for absolute quantification. Automated image analysis algorithms can provide unbiased quantification of signal intensity across defined anatomical regions.

When analyzing developmental changes, consider using temporal cohorts spanning key developmental periods, particularly focusing on critical windows for synaptogenesis and myelination where ANKS1B may play important roles . For comparing wild-type versus ANDS models, use littermate controls and ensure blinded analysis to prevent bias.

Table 1: Recommended Quantification Methods for ANKS1B Across Brain Regions

How might new antibody engineering approaches improve ANKS1B detection specificity and sensitivity?

Emerging antibody engineering technologies offer promising approaches to enhance ANKS1B detection. Recombinant antibody fragments like single-chain variable fragments (scFvs) and nanobodies derived from camelid antibodies provide improved tissue penetration and reduced background compared to conventional antibodies. These smaller molecules can access epitopes in densely packed postsynaptic densities where ANKS1B localizes .

CRISPR-based epitope tagging allows endogenous ANKS1B to be tagged with small epitopes recognized by highly specific antibodies, enabling detection without relying on ANKS1B antibodies directly. This approach preserves native expression levels and localization patterns. For multiplexed detection, DNA-barcoded antibodies enable simultaneous detection of ANKS1B alongside dozens of other proteins in the same sample, allowing comprehensive analysis of synaptic protein networks.

Proximity ligation assays using ANKS1B antibodies can detect specific protein-protein interactions with single-molecule sensitivity, revealing ANKS1B's binding partners like KRIT1, amyloid-beta protein, and EphA receptors in their native context . For challenging applications, antibody affinity maturation through yeast or phage display can generate higher-affinity ANKS1B antibodies, while computational epitope prediction can guide antibody development targeting unique ANKS1B regions to minimize cross-reactivity with related proteins like ANKS1A.

When implementing these advanced approaches, researchers should validate new antibody formats against traditional antibodies to ensure comparable specificity while documenting improved performance metrics.

What emerging methodologies might combine ANKS1B antibodies with spatial transcriptomics to better understand regional brain pathology in ANDS?

Integrating ANKS1B antibodies with spatial transcriptomics offers transformative approaches for understanding ANDS pathology. Combined immunofluorescence and spatial transcriptomics techniques like Visium (10x Genomics) allow correlation of ANKS1B protein distribution with comprehensive transcriptional profiles across intact brain sections. This reveals relationships between ANKS1B protein levels and gene expression networks in specific anatomical contexts.

More advanced approaches like MERFISH (Multiplexed Error-Robust Fluorescence In Situ Hybridization) combined with immunofluorescence using ANKS1B antibodies enable simultaneous visualization of ANKS1B protein and hundreds of mRNA species at subcellular resolution. This approach can identify transcriptional signatures in cells expressing abnormal levels of ANKS1B in ANDS models .

For longitudinal studies, clearing techniques like CLARITY paired with ANKS1B immunolabeling and in situ RNA detection allow whole-brain analysis of protein-RNA relationships across development in ANDS models. Computational integration of these multimodal datasets requires specialized approaches—manifold alignment techniques can identify correlations between protein localization and gene expression patterns, while trajectory inference algorithms can reconstruct developmental abnormalities in ANDS.

When designing these studies, researchers should establish analysis pipelines capable of handling these complex multimodal datasets and consider power calculations to determine appropriate sample sizes for detecting subtle regional differences in ANKS1B expression patterns and associated transcriptional changes.

How can ANKS1B antibodies be integrated into high-content screening approaches for potential therapeutic compounds targeting ANDS?

Integrating ANKS1B antibodies into high-content screening offers powerful approaches for therapeutic discovery for ANDS. Cellular models derived from patient iPSCs (induced pluripotent stem cells) or CRISPR-engineered cell lines with ANKS1B mutations provide disease-relevant screening platforms. High-content imaging using ANKS1B antibodies alongside markers for oligodendrocyte maturation (like MBP) or neuronal function can assess compound effects on multiple cellular phenotypes simultaneously .

Automated immunofluorescence analysis can quantify changes in ANKS1B expression, localization, and post-translational modifications in response to thousands of compounds. Prioritize endpoints with direct relevance to ANDS pathology, such as oligodendrocyte maturation, myelination, or synaptic ANKS1B localization .

Multiplexed assays combining ANKS1B antibodies with functional readouts offer greater insight—for example, combining ANKS1B staining with calcium imaging can reveal compounds that restore both protein levels and neuronal activity patterns. For target validation, include positive control compounds known to affect pathways involving ANKS1B, such as Rac1 modulators given the Rac1 dysfunction observed in Anks1b-deficient models .

Table 2: High-Content Screening Assay Design for ANKS1B-Targeted Therapeutics

The most promising compounds should advance to validation in more complex models, such as brain organoids or Anks1b-deficient mice, with detailed assessment of target engagement and efficacy against ANDS-related phenotypes.