ANKRD10 Antibody

What is ANKRD10 and what is its biological significance?

ANKRD10 is a member of the ankyrin repeat domain-containing protein family characterized by multiple ankyrin repeats in its structure. These ankyrin repeats facilitate protein-protein interactions, suggesting ANKRD10 functions primarily as a scaffolding protein. Similar to other ankyrin repeat-containing proteins, ANKRD10 likely contributes significantly to cellular processes including signal transduction and cell cycle regulation . The protein's structural characteristics suggest it interacts with multiple cellular proteins, potentially serving as a scaffold to organize protein complexes. While less extensively studied than some ankyrin repeat family members, understanding ANKRD10's function is important for comprehending cellular regulatory networks and potential therapeutic targets.

What types of ANKRD10 antibodies are currently available for research?

Several types of ANKRD10 antibodies are available for research applications, with variations in host species, clonality, target regions, and conjugations. The most common are rabbit polyclonal antibodies targeting different regions of the ANKRD10 protein, including the C-terminal region (AA 322-371) and middle regions (AA 100-250) . These antibodies are available in various formats including:

• Unconjugated antibodies for general applications

• Fluorescently-labeled (FITC) for direct immunofluorescence

• Enzyme-conjugated (HRP) for enhanced detection sensitivity

• Biotin-conjugated for amplification systems and multiplex applications

While polyclonal antibodies represent the majority of available reagents, some monoclonal options may be available for specialized applications requiring higher specificity for particular epitopes.

What are the validated applications for ANKRD10 antibodies?

ANKRD10 antibodies have been validated for multiple research applications with varying levels of optimization:

For all applications, optimization of antibody concentration is recommended as performance may vary based on sample type, preparation method, and detection system .

How should ANKRD10 antibodies be stored and handled for optimal performance?

Proper storage and handling of ANKRD10 antibodies are critical for maintaining reactivity and specificity. Most ANKRD10 antibodies are supplied in liquid format with stabilizers such as PBS buffer containing 0.09% sodium azide and 2% sucrose . Recommended storage and handling practices include:

• Store at -20°C for long-term preservation

• For short-term use (up to 1 week), store at 2-8°C

• Avoid repeated freeze-thaw cycles that can denature antibody proteins

• Work with small aliquots to minimize freeze-thaw cycles

• Handle with appropriate precautions, particularly for antibodies containing sodium azide, which is toxic

Before use, allow antibodies to equilibrate to room temperature and centrifuge briefly to collect liquid at the bottom of the tube. For diluted working solutions, prepare fresh whenever possible to maintain optimal binding affinity.

What methods are recommended for validating ANKRD10 antibody specificity?

Validating antibody specificity is critical for generating reliable research findings. For ANKRD10 antibodies, a comprehensive validation approach should include:

• Knockout/knockdown controls: Compare signal between wild-type samples and those with ANKRD10 knocked down (siRNA) or knocked out (CRISPR/Cas9). This methodology has been successfully applied with related ankyrin repeat proteins like ANKRD1 .

• Peptide competition assays: Pre-incubate antibody with excess immunizing peptide before application to sample. Specific signal should be significantly reduced or eliminated.

• Cross-validation with multiple antibodies: Use antibodies targeting different epitopes of ANKRD10 to confirm consistent localization patterns and expression levels.

• Protein expression systems: Test antibody against recombinant ANKRD10 protein alongside negative controls.

• Mass spectrometry validation: Immunoprecipitate ANKRD10 from cell lysates and confirm identity by mass spectrometry to ensure antibody is capturing the intended target.

The combination of these approaches provides robust validation of antibody specificity before proceeding with experimental applications.

How can researchers optimize ANKRD10 immunoprecipitation for protein interaction studies?

Immunoprecipitation (IP) is an essential technique for studying ANKRD10 protein-protein interactions, as demonstrated with related ankyrin repeat proteins . For successful ANKRD10 IP experiments:

• Lysis buffer selection: Use IP-compatible lysis buffers that maintain protein interactions while efficiently extracting ANKRD10. A standard starting point is a buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1% NP-40, with protease and phosphatase inhibitors.

• Subcellular fractionation: Consider preparing nuclear and cytoplasmic extracts separately, as ANKRD10 may have different interaction partners in different cellular compartments, similar to ANKRD1 .

• Antibody selection and immobilization:

  • For primary IP: Use 2-5 μg of ANKRD10 antibody per 500 μg of protein lysate

  • Pre-clear lysate with protein A/G beads to reduce non-specific binding

  • Consider cross-linking antibody to beads for cleaner results

• Validation approaches:

  • Reverse IP: Immunoprecipitate suspected interaction partners and probe for ANKRD10

  • Proximity ligation assay (PLA): Use for in situ visualization of protein interactions

  • Include appropriate negative controls (IgG control, non-expressing cell lines)

• Detection strategies: Western blot remains the standard detection method, but mass spectrometry can provide unbiased identification of novel interaction partners.

What are the considerations for using ANKRD10 antibodies in immunofluorescence studies?

Immunofluorescence microscopy is valuable for determining ANKRD10 subcellular localization. Based on studies of related proteins and validated ANKRD10 antibody applications , researchers should consider:

• Fixation method optimization:

  • 4% paraformaldehyde (10-15 minutes) preserves most epitopes

  • Methanol fixation may be preferred for cytoskeletal studies

  • Test both methods to determine optimal epitope preservation

• Permeabilization considerations:

  • 0.1-0.2% Triton X-100 for nuclear and cytoplasmic proteins

  • 0.05% saponin for membrane-associated proteins

  • Mild permeabilization for potential membrane-associated pools of ANKRD10

• Blocking conditions:

  • 3-5% BSA or normal serum matching secondary antibody host

  • Include 0.1% Triton X-100 in blocking buffer for reduced background

• Antibody concentration:

  • Start with 2 μg/ml dilution as validated for ANKRD10

  • Include appropriate negative controls

• Co-localization studies:

  • Pair with markers for specific cellular compartments (nuclear, cytoplasmic, cytoskeletal)

  • Based on studies with related proteins, consider co-staining with sarcomeric markers

• Image acquisition considerations:

  • Use confocal microscopy for precise localization

  • Acquire z-stacks for three-dimensional localization analysis

  • Include controls for autofluorescence and bleed-through

Published immunofluorescence studies show ANKRD10 in both cytoplasmic and microtubule-associated patterns in U-2 OS cells , suggesting roles in multiple cellular compartments.

How do experimental conditions affect ANKRD10 antibody performance in Western blotting?

Western blotting is one of the most common applications for ANKRD10 antibodies, and several factors can significantly affect performance:

• Sample preparation:

  • Complete denaturation is critical - use SDS and reducing agents

  • ANKRD10 has predicted molecular weight of approximately 42 kDa

  • Consider potential post-translational modifications that may alter mobility

  • Include protease inhibitors to prevent degradation products

• Gel percentage and transfer conditions:

  • 10% Tris-HCl polyacrylamide gels are suitable for resolving ANKRD10

  • Semi-dry transfer works well for mid-sized proteins like ANKRD10

  • Use PVDF membranes for better protein retention and sensitivity

• Blocking and antibody incubation:

  • 5% non-fat dry milk in TBST is standard, but BSA may improve sensitivity

  • Primary antibody concentration: 0.4-1 μg/mL has been validated

  • Overnight incubation at 4°C may improve sensitivity for low abundance samples

• Detection systems:

  • Enhanced chemiluminescence (ECL) provides good sensitivity

  • Fluorescence-based detection offers better quantification potential

  • Consider signal enhancement systems for low-abundance samples

• Validated controls:

  • Positive controls: RT4 and U-251 MG cell lysates have demonstrated detectable ANKRD10 expression

  • Loading controls: β-actin, GAPDH, or α-tubulin depending on experimental context

What approaches are effective for studying ANKRD10 function through knockdown or knockout experiments?

Functional studies of ANKRD10 can be performed through gene silencing or knockout approaches. Based on methodologies applied to related proteins :

• siRNA-mediated knockdown:

  • Design 3-4 siRNA sequences targeting different regions of ANKRD10 mRNA

  • Transfect using appropriate method for cell type (lipofection for most cell lines)

  • Verify knockdown efficiency by Western blot (protein) and qRT-PCR (mRNA)

  • Typical concentrations: 20-50 nM siRNA for 48-72 hours

• shRNA for stable knockdown:

  • Consider lentiviral delivery for hard-to-transfect cells

  • Include selection marker (puromycin, G418) for stable line generation

  • Verify knockdown maintenance over multiple passages

• CRISPR/Cas9 knockout:

  • Design sgRNAs targeting early exons of ANKRD10

  • Consider inducible systems for developmental studies

  • Generate homozygous and heterozygous models to assess dose-dependent effects

  • Complete knockout validation via sequencing and protein expression analysis

• Rescue experiments:

  • Re-express siRNA-resistant ANKRD10 constructs to confirm specificity

  • Consider domain mutants to determine functional regions

• Phenotypic analysis:

  • Based on suspected functions in signal transduction and cell cycle regulation

  • Assess proliferation, cell cycle distribution, and morphology

  • Evaluate effects on potential interaction partners

  • Examine impact on signaling pathways similar to those affected by other ankyrin repeat proteins

Studies of related protein ANKRD1 have successfully used siRNA approaches to demonstrate its role in signaling complexes , suggesting similar approaches would be effective for ANKRD10.

How can researchers investigate ANKRD10 expression patterns in brain and other tissues?

ANKRD10 shows tissue-specific expression patterns that may inform understanding of its function. Based on tissue expression data and methods used for related proteins:

• Transcriptomic analysis:

  • Review RNA-seq datasets from resources like Human Protein Atlas

  • Compare expression across tissues and developmental stages

  • Consider single-cell RNA-seq for cell-type specificity

• Protein-level validation:

  • Immunohistochemistry with validated ANKRD10 antibodies

  • Western blot analysis of tissue lysates with appropriate loading controls

  • Optimize antigen retrieval methods (citrate buffer has been effective)

• Brain region specificity:

  • ANKRD10 expression varies across brain regions

  • Use regional tau specificity scores to quantify differential expression

  • Compare with related ankyrin repeat proteins for pattern similarities

• Developmental regulation:

  • Assess expression changes during development

  • Compare with developmental timing of potential interaction partners

• Disease state alterations:

  • Compare expression between normal and pathological tissues

  • Consider correlation with disease progression markers

The combination of transcriptomic data with protein-level validation provides the most comprehensive view of ANKRD10 expression patterns.

What methodologies can be used to investigate ANKRD10's role in signaling pathways?

Based on research with related ankyrin repeat proteins , several approaches can be used to determine ANKRD10's role in signaling pathways:

• Signaling pathway stimulation and inhibition:

  • Treat cells with pathway agonists (e.g., growth factors, receptor ligands)

  • Use specific pathway inhibitors to determine dependence relationships

  • Monitor ANKRD10 phosphorylation, localization changes, or interaction partners

• Co-immunoprecipitation coupled with phosphorylation analysis:

  • Immunoprecipitate ANKRD10 after pathway stimulation

  • Probe for co-precipitating signaling molecules

  • Assess phosphorylation state of ANKRD10 and binding partners using phospho-specific antibodies

• Proximity-based methods:

  • Proximity ligation assay (PLA) to visualize interactions in situ

  • BioID or APEX2 proximity labeling to identify neighboring proteins

• Functional readouts:

  • Reporter gene assays for transcriptional effects

  • Phenotypic assays specific to the pathway of interest

  • Rescue experiments with constitutively active or dominant negative constructs

• Proteomic approaches:

  • Phosphoproteomic analysis following ANKRD10 manipulation

  • Interaction proteomics to identify pathway components

Similar to ANKRD1's role in the sarcomeric ERK-GATA4 signaling pathway , ANKRD10 may participate in specific signaling complexes that can be elucidated through these approaches.

How should researchers design experiments to investigate tissue-specific functions of ANKRD10?

Investigating tissue-specific functions requires careful experimental design:

• Model selection:

  • Choose cell lines that represent the tissue of interest

  • Consider primary cells for physiological relevance

  • Evaluate organoid models for 3D organization

• Expression validation:

  • Confirm ANKRD10 expression in selected models

  • Compare expression levels to the tissue of interest

  • Consider inducible expression systems for temporal control

• Tissue-specific interaction partners:

  • Perform immunoprecipitation followed by mass spectrometry in different tissues

  • Compare interactomes between tissues to identify unique partners

  • Validate key interactions through orthogonal methods

• Conditional knockout approaches:

  • Use tissue-specific promoters to drive Cre recombinase in floxed models

  • Temporal control can be achieved with inducible systems

  • Compare phenotypes across different tissue-specific knockouts

• Ex vivo and in vivo validation:

  • Tissue explants maintain tissue architecture while allowing manipulation

  • In vivo studies provide systemic context for observed effects

  • Consider both acute (siRNA) and chronic (genetic) interventions

This approach has been successfully applied to study tissue-specific functions of related ankyrin repeat proteins like ANKRD1 in cardiac tissue .

What controls are essential when troubleshooting inconsistent results with ANKRD10 antibodies?

When encountering inconsistent results with ANKRD10 antibodies, systematic troubleshooting with appropriate controls is essential:

• Positive and negative sample controls:

  • Positive: Known ANKRD10-expressing samples (e.g., RT4 or U-251 MG cell lines)

  • Negative: ANKRD10 knockdown/knockout samples

  • Recombinant ANKRD10 protein as absolute positive control

• Antibody controls:

  • Primary antibody omission to assess secondary antibody specificity

  • Isotype control to identify non-specific binding

  • Test multiple ANKRD10 antibodies targeting different epitopes

  • Peptide competition to confirm epitope specificity

• Protocol controls:

  • Include loading controls (β-actin, GAPDH) for Western blots

  • Run molecular weight markers to confirm target band identity

  • Process all samples simultaneously to minimize technical variation

• Sample preparation variables:

  • Test multiple lysis buffers for protein extraction efficiency

  • Compare fresh vs. frozen samples

  • Evaluate different fixatives for immunohistochemistry/immunofluorescence

• Detection system checks:

  • Test multiple secondary antibodies or detection systems

  • Include known antibodies that work well with your detection system

  • Evaluate potential interfering substances in samples

Systematic implementation of these controls can identify sources of variability and improve experimental reproducibility.

How can researchers effectively design quantitative studies of ANKRD10 expression levels?

For accurate quantification of ANKRD10 expression:

• Western blot quantification:

  • Use fluorescence-based detection for wider linear range

  • Include standard curve of recombinant protein when possible

  • Load multiple concentrations to ensure linearity of signal

  • Use total protein normalization (e.g., Stain-Free technology) rather than single housekeeping proteins

  • Apply appropriate statistical analysis for technical and biological replicates

• qRT-PCR for mRNA quantification:

  • Design primers spanning exon junctions to avoid genomic DNA amplification

  • Validate primer efficiency with standard curves

  • Use multiple reference genes selected for stability in your experimental system

  • Apply appropriate normalization method (ΔΔCt, standard curve)

• Immunohistochemistry quantification:

  • Standardize all staining parameters (fixation, antibody concentration)

  • Use automated image analysis to reduce subjective interpretation

  • Include calibration standards on each slide

  • Score sufficient fields/cells for statistical power

• Flow cytometry:

  • Include fluorescence minus one (FMO) controls

  • Use median fluorescence intensity for quantification

  • Standardize instrument settings across experiments

• Absolute quantification approaches:

  • Consider AQUA (Absolute Quantification) peptides for mass spectrometry

  • Digital PCR for absolute copy number determination

What are common pitfalls when working with ANKRD10 antibodies, and how can they be addressed?

Several technical challenges are commonly encountered when working with antibodies against low-abundance proteins like ANKRD10:

• Non-specific binding:

  • Solution: Increase blocking time/concentration

  • Use casein or protein-free blockers if milk/BSA is ineffective

  • Pre-adsorb antibody with tissues/cells lacking ANKRD10

  • Optimize antibody concentration (lower isn't always better)

• Weak or no signal:

  • Solution: Try different epitope retrieval methods for IHC/IF

  • Increase antibody concentration or incubation time

  • Use signal amplification systems (TSA, ABC systems)

  • Check sample preparation for potential protein degradation

• Multiple bands in Western blot:

  • Solution: Verify with ANKRD10 knockdown controls

  • Consider post-translational modifications or alternative splice variants

  • Use more stringent washing conditions to reduce non-specific binding

  • Evaluate the presence of degradation products

• Variability between experiments:

  • Solution: Standardize all protocols precisely

  • Prepare larger antibody aliquots to reduce freeze-thaw cycles

  • Use automated systems where possible to reduce operator variation

  • Include internal standards across experiments

• Background in immunostaining:

  • Solution: Optimize blocking conditions

  • Include detergents in wash buffers

  • Consider autofluorescence quenching for certain tissues

  • Use Fab fragments or monovalent antibodies to reduce non-specific binding

Careful validation and optimization can address most of these common challenges.

How can researchers differentiate between ANKRD10 and other ankyrin repeat proteins in their studies?

Distinguishing ANKRD10 from related ankyrin repeat proteins requires careful experimental design:

• Antibody selection:

  • Choose antibodies targeting unique regions outside conserved ankyrin repeats

  • Validate specificity against recombinant proteins of multiple family members

  • Consider raised-together/purified-separately antibody sets for comparative studies

• Western blot differentiation:

  • Leverage size differences between family members

  • Use high-resolution gels to separate similarly sized proteins

  • Include positive controls for each family member of interest

• Specificity validation:

  • Perform peptide competition with specific peptides from each family member

  • Test antibody against overexpressed tagged constructs of various family members

  • Verify with knockdown/knockout controls for each protein

• mRNA-level discrimination:

  • Design primers targeting unique regions

  • Validate specificity using cloned standards

  • Consider RNAscope or similar technologies for in situ discrimination

• Mass spectrometry approaches:

  • Identify unique peptides for each family member

  • Use targeted approaches (MRM/PRM) for specific quantification

  • Apply SILAC labeling for comparative studies

Related ankyrin repeat proteins like ANKRD1 share structural features with ANKRD10 , making careful discrimination essential for accurate interpretation of results.

What are the best approaches for investigating post-translational modifications of ANKRD10?

Post-translational modifications (PTMs) can significantly impact ANKRD10 function. Based on studies of related proteins , effective approaches include:

• Phosphorylation analysis:

  • Immunoprecipitate ANKRD10 followed by phospho-specific antibody detection

  • Use phosphatase treatments as controls

  • Apply phosphoproteomic mass spectrometry for site identification

  • Consider Phos-tag gels for mobility shift detection

  • Use kinase inhibitors to identify responsible signaling pathways

• Ubiquitination and SUMOylation:

  • Co-IP with tagged ubiquitin/SUMO constructs

  • Use deubiquitinating enzyme inhibitors to stabilize modifications

  • Apply TUBEs (Tandem Ubiquitin Binding Entities) for enrichment

  • Investigate proteasome involvement with inhibitors like MG132

• Other modifications:

  • Acetylation: IP followed by anti-acetyl-lysine antibody detection

  • Methylation: Methylation-specific antibodies or mass spectrometry

  • Glycosylation: Lectin-based detection or glycosidase treatments

• Functional consequences:

  • Generate modification site mutants (e.g., S→A for phosphorylation)

  • Assess effects on localization, stability, and interactions

  • Use phosphomimetic mutations (S→D/E) for functional studies

• Stimulus-dependent changes:

  • Compare modifications under different cellular conditions

  • Track modification kinetics after stimulus application

  • Correlate with functional outcomes

Studies of related proteins suggest phosphorylation may regulate ANKRD10 localization and interaction partnerships, similar to observations with ANKRD1 .

How might ANKRD10 be involved in disease processes, and what methodologies can investigate these connections?

While specific disease associations for ANKRD10 are still emerging, several approaches can investigate potential roles:

• Expression correlation with disease states:

  • Compare ANKRD10 levels between normal and diseased tissues

  • Analyze public databases for expression changes in disease cohorts

  • Investigate associations with disease progression or outcomes

• Functional studies in disease models:

  • Manipulate ANKRD10 levels in cellular disease models

  • Assess effects on disease-relevant phenotypes

  • Evaluate impact on established disease pathways

• Genetic association approaches:

  • Analyze GWAS data for ANKRD10 locus associations

  • Investigate coding variants for functional consequences

  • Consider regulatory variants affecting expression

• Protein interaction context:

  • Identify ANKRD10 interaction partners implicated in disease

  • Investigate whether these interactions are altered in disease states

  • Determine if ANKRD10 modifies activity of disease-associated proteins

• Therapeutic targeting assessment:

  • Evaluate ANKRD10 as a potential biomarker

  • Consider protein-protein interaction disruption approaches

  • Assess whether ANKRD10 modulation affects response to existing therapies

The brain expression pattern of ANKRD10 suggests potential neurological roles that warrant investigation, while its structural similarities to ANKRD1 point to possible involvement in signaling pathways relevant to multiple diseases.

What new technologies are emerging for studying ANKRD10 and other challenging low-abundance proteins?

Several cutting-edge technologies offer advantages for studying challenging proteins like ANKRD10:

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM, SIM) for detailed localization

  • Live-cell imaging with split-fluorescent proteins for interaction dynamics

  • Correlative light-electron microscopy for ultrastructural context

  • Expansion microscopy for improved spatial resolution

• Single-cell technologies:

  • Single-cell proteomics for cell-to-cell variation

  • Single-cell Western blotting for protein heterogeneity

  • Mass cytometry (CyTOF) for multiparameter analysis

• CRISPR technologies:

  • CRISPRa/i for endogenous gene modulation

  • Base editing for specific mutation introduction

  • CRISPR screens for functional pathway mapping

  • Prime editing for precise genetic modification

• Proximity labeling approaches:

  • BioID, TurboID, or APEX2 for identifying neighboring proteins

  • Split-BioID for conditional proximity labeling

  • Organelle-specific proximity labeling

• Protein-protein interaction visualization:

  • FRET/BRET for dynamic interaction monitoring

  • Three-hybrid systems for complex formation analysis

  • Advanced PLA methods for subcellular resolution of interactions

These emerging technologies provide unprecedented opportunities to study ANKRD10 function, interactions, and dynamics with greater sensitivity and resolution than traditional approaches.