ANKS6 Antibody

What is ANKS6 and why is it important in research?

ANKS6, or Ankyrin Repeat and Sterile alpha Motif Domain Containing 6, is a protein with significant roles in cellular function and disease pathology. In humans, the canonical ANKS6 protein consists of 871 amino acid residues with a molecular mass of 92.2 kDa . It localizes primarily in cell projections and cytoplasm and exists in up to three different isoforms . ANKS6 is widely expressed across various tissue types and plays a crucial role in renal function .

Research interest in ANKS6 stems from its association with nephronophthisis, a genetic kidney disease characterized by renal cysts and progressive kidney failure . ANKS6 serves as a central component of a nephronophthisis module that links several NPHP-associated proteins, including NEK8, INVS, and NPHP3 . This network controls normal renal and cardiovascular development, making ANKS6 antibodies essential tools for studying these developmental processes and associated pathologies.

What are the common synonyms and orthologs for ANKS6?

When searching literature or databases for ANKS6-related information, researchers should be aware of several synonyms:

• SAM domain-containing protein 6

• Ankyrin repeat domain 14

• samCystin

• Ankyrin repeat and SAM domain-containing protein 6

ANKS6 is highly conserved across species, with orthologs reported in:

• Mouse (Mus musculus)

• Rat (Rattus norvegicus)

• Bovine (Bos taurus)

• Frog (Xenopus species)

• Chimpanzee (Pan troglodytes)

• Chicken (Gallus gallus)

These orthologs enable comparative studies across different model organisms to understand conserved functions of ANKS6.

What are the primary applications for ANKS6 antibodies in research?

ANKS6 antibodies serve multiple experimental applications in research settings:

The choice of application depends on the specific research question, with immunohistochemistry being particularly prevalent in ANKS6 research due to its importance in understanding tissue expression patterns .

How does ANKS6 contribute to ciliary function?

ANKS6 plays a critical role in ciliary function through its interactions with other ciliary proteins. Research has shown that ANKS6 localizes to the proximal segment of cilia in a manner dependent on INVS (Inversin) . In Invs-depleted IMCD3 cells, endogenous Anks6 is lost from the proximal segment of cilia without affecting total Anks6 protein levels .

ANKS6 functions as an organizer of a distinct NPHP (nephronophthisis) protein module by linking INVS and NPHP3 to NEK8 . This module is essential for normal ciliary function and signaling. Disruption of this complex through mutations or altered protein interactions can lead to ciliopathies characterized by renal and cardiovascular abnormalities .

How do post-translational modifications affect ANKS6 function and detection with antibodies?

ANKS6 undergoes hydroxylation, a critical post-translational modification that affects its protein-protein interactions. Mass spectrometry has detected hydroxylated peptides at asparagine N129 of Anks6, mediated by the asparaginyl-hydroxylase HIF1AN (Factor Inhibiting HIF, FIH) . This oxygen-dependent hydroxylation appears to alter the composition of the ANKS6 complex.

When designing experiments with ANKS6 antibodies, researchers should consider:

• Antibody epitope location: Antibodies targeting regions near modification sites may show differential binding depending on the modification state

• Experimental conditions: Hypoxic conditions may alter HIF1AN-mediated hydroxylation of ANKS6

• Protein interactions: Mutation of hydroxylation sites in ANKS6 results in decreased binding to NEK8

For studies focusing on ANKS6 interactions, researchers should validate whether their antibodies can detect both modified and unmodified forms of the protein, as this may significantly impact experimental outcomes and interpretation.

What are the optimal experimental conditions for detecting ANKS6 in different subcellular compartments?

Detecting ANKS6 in different subcellular compartments requires careful optimization of experimental conditions:

For ciliary localization studies:

• Cell fixation method: Paraformaldehyde (4%) preservation of ciliary structures is critical

• Permeabilization: Mild detergents (0.1-0.2% Triton X-100) maintain ciliary architecture

• Co-staining markers: Include acetylated tubulin to mark cilia and verify ANKS6 localization to the proximal ciliary segment

• INVS status: Consider that ANKS6 localization depends on INVS, as ANKS6 is lost from proximal cilia in INVS-depleted cells

For cytoplasmic detection:

• Use antibodies targeting central regions (e.g., AA 505-534) of ANKS6

• Consider subcellular fractionation before Western blotting to enrich cytoplasmic fractions

For optimal results, researchers should validate their antibodies in both wild-type tissues/cells and those with altered ANKS6 expression or mutation to confirm specificity.

How do ANKS6 mutations affect epitope recognition by different antibodies?

ANKS6 mutations can significantly impact antibody epitope recognition, particularly missense mutations that alter protein conformation or post-translational modifications:

The Gln441Arg mutation (identified in patient B7397) presents a model case: while this mutation didn't alter interaction with other NPHP proteins or ciliary localization , it failed to rescue the renal phenotype in Xenopus models . This suggests that:

• Structurally conserved mutations may maintain epitope recognition by antibodies targeting distant regions

• Functional changes may occur despite preserved antibody binding

• Researchers should validate antibodies against both wild-type and mutant proteins

When studying patient samples with known ANKS6 mutations, researchers should:

• Choose antibodies targeting epitopes distant from mutation sites

• Consider using multiple antibodies targeting different regions of ANKS6

• Include appropriate positive and negative controls for validation

This approach ensures reliable detection regardless of potential conformational changes induced by mutations.

What is the significance of the ANKS6-ANKS3-Bicc1 interaction network in experimental design?

The interaction between ANKS6, ANKS3, and Bicc1 represents a complex regulatory network with significant implications for experimental design:

Recent structure-function analysis reveals that ANKS3 can either inhibit or promote the binding of target mRNAs depending on its conformation and regulation by ANKS6 . Specifically:

• The C-terminal coiled-coil domain of ANKS3 interacts with Bicc1

• This interaction inhibits binding of target mRNAs

• ANKS6 regulates this inhibitory effect by modulating ANKS3 conformation

When studying any component of this network, researchers should consider:

• The multivalent nature of these protein interactions

• The dynamic regulation of RNA binding

• The potential impact on ribonucleoparticle (RNP) formation

• The connection to laterality defects and other ciliopathies

Experimental approaches might include co-immunoprecipitation with antibodies against different components, in vitro reconstitution of complexes, and assessment of RNA binding in the presence of various protein combinations.

What are the best practices for validating ANKS6 antibody specificity?

Validating ANKS6 antibody specificity is crucial for generating reliable research results:

• Genetic validation approaches:

  • ANKS6 knockout/knockdown controls: Use CRISPR/Cas9-mediated knockout or siRNA knockdown of ANKS6 to confirm antibody specificity

  • Overexpression systems: Test antibody recognition of tagged recombinant ANKS6 proteins

• Biochemical validation:

  • Western blot analysis: Confirm detection of bands at the expected molecular weight (92.2 kDa for canonical human ANKS6)

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

  • Cross-reactivity assessment: Test against closely related proteins like ANKS3

• Tissue validation:

  • Compare staining patterns with known ANKS6 expression profiles

  • Verify subcellular localization in cell projections and cytoplasm

  • Check detection in tissues with established ANKS6 expression levels

Including proper validation steps prevents misinterpretation of data and ensures the reliability of experimental findings.

How should researchers select the appropriate ANKS6 antibody for specific applications?

Selecting the optimal ANKS6 antibody requires careful consideration of several factors:

• Epitope targeting:

  • For studying protein interactions: Choose antibodies targeting regions away from interaction domains

  • For post-translational modifications: Select antibodies that can discriminate between modified and unmodified states

  • For isoform-specific detection: Use antibodies targeting unique regions of specific isoforms

• Application-specific considerations:

  • For Western blot: Antibodies targeting AA 505-534 from the central region have shown good results

  • For immunohistochemistry: Consider whether the epitope is preserved after fixation procedures

  • For immunoprecipitation: Verify that the antibody doesn't interfere with protein-protein interactions of interest

• Species reactivity:

  • Confirm cross-reactivity with the model organism being studied

  • Consider evolutionary conservation of the epitope region across species

Researchers should review available validation data and published literature using specific antibodies before making their selection.

What experimental controls are essential when studying ANKS6 interactions with NPHP proteins?

When investigating ANKS6 interactions with other NPHP proteins, several critical controls must be included:

• Input controls:

  • Expression level verification: Confirm equal expression of all proteins under study

  • Subcellular localization: Verify proper localization of proteins before interaction studies

  • Protein solubility: Ensure extraction conditions maintain native protein conformations

• Interaction-specific controls:

  • Negative controls: Include non-interacting proteins with similar properties

  • Competition assays: Use excess untagged protein to compete with tagged protein

  • Domain deletion/mutation: Test specific domains/residues required for interaction

  • HIF1AN activity: Consider the role of hydroxylation in mediating interactions

• Validation across methods:

  • Reciprocal co-immunoprecipitation experiments

  • Proximity ligation assays for in situ verification

  • In vitro binding assays with purified components

These controls help distinguish specific interactions from experimental artifacts and provide robust evidence for physical associations between ANKS6 and other NPHP module components.

How can ANKS6 antibodies contribute to nephronophthisis research?

ANKS6 antibodies play a crucial role in nephronophthisis research by enabling multiple investigative approaches:

• Pathological assessment:

  • Immunohistochemical staining of patient kidney biopsies

  • Evaluation of ANKS6 expression and localization in cystic versus normal tissue

  • Comparison between wild-type and mutant ANKS6 localization patterns

• Molecular mechanism studies:

  • Investigation of ANKS6 module assembly in disease models

  • Analysis of how disease-causing mutations affect ANKS6 interactions with NEK8, INVS, and NPHP3

  • Assessment of ciliary localization defects in patient-derived cells

• Diagnostic development:

  • Identifying ANKS6 mislocalization as a potential biomarker

  • Developing immunoassays for detecting mutant forms

  • Creating antibody-based screening methods for high-risk populations

By enabling these applications, ANKS6 antibodies facilitate both basic understanding of disease mechanisms and translational advances in nephronophthisis research.

What insights can ANKS6 antibodies provide about cardiovascular defects associated with ANKS6 mutations?

ANKS6 mutations have been linked to cardiovascular abnormalities, including aortic stenosis, pulmonary stenosis, and obstructive cardiomyopathy . ANKS6 antibodies can provide valuable insights into these conditions:

• Developmental studies:

  • Tracking ANKS6 expression during cardiac development

  • Identifying critical time points when ANKS6 function affects heart morphogenesis

  • Comparing normal versus aberrant cardiac developmental trajectories

• Tissue-specific analyses:

  • Characterizing ANKS6 expression patterns in different heart regions

  • Correlating mislocalization with specific structural defects

  • Investigating cell-type specific effects in cardiac tissues

• Mechanistic investigations:

  • Determining whether cardiovascular phenotypes result from primary ANKS6 dysfunction or secondary effects

  • Exploring relationships between ANKS6 and other proteins linked to cardiac development

  • Examining potential therapeutic targets within the ANKS6 pathway

These investigations may clarify why specific ANKS6 mutations (like those in patients A3114, B6794, A649, and NPH316) manifest as cardiovascular defects rather than solely renal abnormalities .

How can researchers use ANKS6 antibodies to investigate the role of oxygen sensing in ciliopathies?

The discovery that ANKS6 interacts with HIF1AN (an oxygen sensor that hydroxylates ANKS6) opens new research directions linking oxygen sensing to ciliopathies:

• Hydroxylation-specific detection:

  • Developing antibodies specifically recognizing hydroxylated ANKS6 at asparagine N129

  • Mapping hydroxylation patterns across tissues and developmental stages

  • Comparing hydroxylation status between normal and disease states

• Hypoxia response studies:

  • Investigating how oxygen levels affect ANKS6 complex formation

  • Determining whether hypoxia-induced changes in ANKS6 hydroxylation contribute to disease

  • Exploring potential therapeutic interventions targeting this pathway

• Mechanistic investigations:

  • Analyzing how HIF1AN facilitates ANKS6/INVS/NPHP3 module formation

  • Studying how hydroxylation alters protein binding capacities

  • Examining the evolutionary conservation of this regulatory mechanism

These approaches could reveal novel connections between environmental oxygen sensing and developmental disorders, potentially identifying new therapeutic strategies for ciliopathies.

How can researchers address non-specific binding issues with ANKS6 antibodies?

Non-specific binding is a common challenge when working with ANKS6 antibodies. Researchers can implement several strategies to minimize this issue:

• Blocking optimization:

  • Test different blocking agents (BSA, milk, normal serum)

  • Increase blocking concentration (3-5% instead of standard 1%)

  • Extend blocking time (2 hours to overnight)

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Perform titration experiments to determine optimal concentration

  • Consider using higher dilutions than manufacturer recommendations

  • Extend primary antibody incubation time at lower concentrations

• Sample-specific considerations:

  • For tissues with high endogenous biotin, use biotin-blocking systems

  • Pre-adsorb antibodies with tissues from knockout models if available

  • Perform antigen retrieval optimization for fixed tissues

These approaches should be systematically tested and documented to establish reliable protocols for specific experimental systems.

What strategies can overcome detection challenges in tissues with low ANKS6 expression?

Detecting ANKS6 in tissues with low expression levels requires specialized approaches:

• Signal amplification methods:

  • Tyramide signal amplification (TSA) for immunohistochemistry/immunofluorescence

  • Enhanced chemiluminescence substrates for Western blotting

  • Biotin-streptavidin amplification systems

• Sample enrichment techniques:

  • Subcellular fractionation to concentrate relevant compartments

  • Immunoprecipitation before Western blotting

  • Proximity ligation assay for detecting protein interactions with higher sensitivity

• Technical considerations:

  • Extended exposure times for Western blots

  • Digital image enhancement with appropriate controls

  • Use of highly sensitive cameras for immunofluorescence

These methodological adaptations can significantly improve detection of low-abundance ANKS6 while maintaining experimental rigor and reproducibility.

How should researchers interpret discrepancies in ANKS6 detection between different antibodies?

Discrepancies between different ANKS6 antibodies are not uncommon and may reflect biological phenomena rather than technical artifacts:

• Possible biological explanations:

  • Isoform-specific detection: Different antibodies may recognize distinct ANKS6 isoforms

  • Post-translational modifications: Some epitopes may be masked by modifications

  • Protein interactions: Certain antibodies may be unable to access epitopes in protein complexes

  • Conformational states: ANKS6 may adopt different conformations in various contexts

• Verification approaches:

  • Use multiple antibodies targeting different regions

  • Compare monoclonal and polyclonal antibodies

  • Validate with genetic approaches (overexpression, knockdown)

  • Perform epitope mapping studies

• Reporting considerations:

  • Document all antibodies used (catalog numbers, dilutions)

  • Specify epitope regions and detection methods

  • Acknowledge limitations in interpretation

  • Consider the biological significance of discrepancies

By treating discrepancies as potentially informative rather than merely problematic, researchers can gain deeper insights into ANKS6 biology and function.