ANKRD23 Antibody

What is ANKRD23 and what cellular processes is it involved in?

ANKRD23 is a member of the muscle ankyrin repeat protein (MARP) family that functions as a transcriptional regulator localized primarily to the nucleus. The protein contains four tandem ankyrin-like repeats and is involved in various cellular processes including:

• Energy metabolism regulation

• Response to mechanical stimulus

• Cell growth and differentiation

• Apoptotic processes

• Recovery following starvation (its expression is induced during this period)

At the molecular level, ANKRD23 interacts with the cytoskeleton, particularly through titin binding, and may serve as a molecular link between myofibrillar stretch-induced signaling pathways and muscle gene expression. This suggests its importance in mechano-transduction and adaptation to mechanical stress .

What applications are ANKRD23 antibodies validated for?

ANKRD23 antibodies have been validated for multiple experimental applications, with varying recommended dilutions depending on the specific antibody and application:

These applications have been validated using specific tissues, particularly muscle tissues where ANKRD23 is predominantly expressed. For Western blotting, positive detection has been confirmed in mouse skeletal muscle tissue with an observed band size of approximately 35 kDa .

How should I validate an ANKRD23 antibody before using it in my experiments?

Proper antibody validation is critical for ensuring reliable experimental results. For ANKRD23 antibodies, validation should include:

• Positive control testing: Use tissues known to express ANKRD23, particularly skeletal and cardiac muscle samples. Mouse skeletal muscle tissue has been confirmed as a positive control for Western blotting .

• Multiple antibody comparison: When possible, use two antibodies targeting different epitopes of ANKRD23. As demonstrated in multiple studies, using antibodies that span different regions of the protein (NH₂-terminal and full-length) provides stronger validation .

• Recombinant protein validation: Test antibody specificity using human ANKRD23 recombinant pure protein as a positive control .

• Cross-reactivity assessment: Test in both human and animal tissue extracts (such as human skeletal muscle and heart extract, and rat heart samples) to confirm species reactivity .

• Negative controls: Include tissues with minimal ANKRD23 expression or use siRNA knockdown approaches where appropriate.

This multi-faceted validation approach ensures that your antibody is specific and reliable for your particular application and experimental system .

How should I design experiments to study ANKRD23 expression in different muscle fiber types?

When investigating ANKRD23 expression across different muscle fiber types, a comprehensive approach requires careful experimental design:

• Sample preparation: Collect both whole muscle homogenates and single muscle fiber segments to compare bulk tissue expression with fiber-specific expression patterns.

• Fiber typing: Use established myosin heavy chain isoform antibodies (anti-MHCI, anti-MHCIIa, anti-MHCIIx) to identify specific fiber types within your samples. These can be used at dilutions around 1:200 for immunohistochemistry .

• Co-localization studies: Perform double immunostaining with ANKRD23 antibodies and fiber-type markers to directly correlate expression with specific fiber types.

• Quantitative assessment: Use densitometry analysis of Western blots to quantify expression levels, normalizing to appropriate loading controls such as GAPDH (1:10,000 dilution recommended) .

• Controls: Include both positive controls (tissues known to express ANKRD23) and negative controls (tissues with minimal expression) in each experiment.

This approach allows for robust characterization of ANKRD23 expression patterns across different muscle fiber types, providing insights into its potential functional specialization .

What considerations should be made when studying ANKRD23 in the context of mechanical stress response?

ANKRD23 is implicated in mechanical stress response pathways, requiring specific experimental considerations:

• Mechanical stimulation models: Utilize appropriate in vitro or in vivo models of mechanical stimulation, such as:

  • Stretch application to cultured myoblasts or myotubes

  • Eccentric exercise protocols in animal models

  • Resistance training in human subjects

• Temporal expression analysis: Monitor ANKRD23 expression at multiple time points post-stimulation (immediate, 6h, 24h, 48h) to capture dynamic regulation.

• Phosphorylation status: Consider examining both total ANKRD23 and its phosphorylated forms, particularly at the serine 99 residue which may be important for activity regulation. Specific antibodies against phosphorylated forms (e.g., anti-pAnkrd2-Ser99) can be used at 1:250 dilution .

• Subcellular localization: Assess potential translocation between cytoplasmic and nuclear compartments in response to mechanical stimuli using subcellular fractionation or immunofluorescence.

• Pathway interactions: Examine interactions with known mechanosensitive pathways by co-immunoprecipitation or proximity ligation assays.

These approaches allow for comprehensive characterization of ANKRD23's role in mechanotransduction, potentially revealing new insights into muscle adaptation to mechanical stress .

What are the common issues with Western blot detection of ANKRD23 and how can they be resolved?

Western blot detection of ANKRD23 can present several challenges that require specific troubleshooting approaches:

• Multiple bands: ANKRD23 may appear as multiple bands (predicted at 30 and 35 kDa) due to potential isoforms or post-translational modifications. To address this:

  • Compare band patterns with recombinant ANKRD23 protein

  • Use isoform-specific antibodies when available

  • Consider phosphatase treatment to eliminate phosphorylation-dependent band shifts

• Weak signal: If experiencing weak detection:

  • Increase antibody concentration (start with 1:500 and adjust as needed)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use enhanced chemiluminescence substrate (like SuperSignal West Femto)

  • Apply Miser antibody extender solution to nitrocellulose membranes

• High background: To reduce non-specific binding:

  • Optimize blocking (5% skim milk in TBST is effective)

  • Increase washing steps (at least 3-5 washes of 5-10 minutes each)

  • Decrease secondary antibody concentration

  • Use fresh buffers and reagents

• Loading control selection: When studying muscle tissue samples with varied fiber composition, traditional loading controls may vary. Consider:

  • Total protein normalization using stain-free technology

  • Multiple loading controls (GAPDH at 1:10,000 and topoisomerase IIβ at 1:2,000)

These optimizations will help ensure reliable and reproducible detection of ANKRD23 in Western blot applications.

How can I optimize immunohistochemical detection of ANKRD23 in different tissue types?

Optimizing immunohistochemical detection of ANKRD23 requires tissue-specific considerations:

• Fixation protocol optimization:

  • For muscle tissue: 4% paraformaldehyde for 10-15 minutes typically preserves structure while maintaining antigenicity

  • For tissues with high connective tissue content: Consider additional antigen retrieval steps

• Antibody dilution optimization:

  • Start with manufacturer-recommended range (1:20-1:200)

  • Perform titration experiments with serial dilutions to determine optimal signal-to-noise ratio

  • Different tissue types may require different optimal dilutions

• Antigen retrieval methods:

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

  • Enzymatic retrieval for heavily fixed tissues

  • Test multiple retrieval methods to determine optimal conditions for your specific tissue

• Signal enhancement:

  • Consider tyramide signal amplification for low-abundance expression

  • Biotin-streptavidin systems can enhance detection sensitivity

  • Balance signal enhancement with potential background increase

• Background reduction:

  • Include tissue-matched blocking serum (5-10%)

  • Add 0.1-0.3% Triton X-100 for better antibody penetration

  • Include avidin/biotin blocking steps if using biotin-based detection systems

These optimization steps should be systematically tested and documented to establish a reliable protocol for your specific tissue and application .

How can ANKRD23 antibodies be utilized to study its role in energy metabolism pathways?

ANKRD23 (DARP) has been implicated in energy metabolism regulation, and antibodies can be used to investigate this function through several approaches:

• Metabolic stress models:

  • Starvation-refeeding experiments (ANKRD23 expression is induced during recovery following starvation)

  • Exercise or training interventions

  • Diabetes models (given its alternative name as Diabetes-related ankyrin repeat protein)

• Co-immunoprecipitation studies:

  • Use ANKRD23 antibodies (1:500-1:1000 dilution) to pull down protein complexes

  • Identify novel interaction partners involved in metabolic regulation

  • Confirm interactions with known metabolic regulators

• Phosphorylation state analysis:

  • Examine how metabolic signals affect ANKRD23 phosphorylation

  • Use phospho-specific antibodies (like those targeting Ser99) alongside total ANKRD23 antibodies

  • Correlate phosphorylation changes with metabolic parameters

• Subcellular localization changes:

  • Monitor potential translocation between cellular compartments during metabolic challenges

  • Combine with metabolic inhibitor treatments to identify pathway dependencies

• Signaling pathway integration:

  • Examine relationships with the Akt pathway using anti-Akt (1:500) and anti-p-Akt2(Ser474) (1:1000) antibodies

  • Investigate cross-talk with other energy-sensing pathways

These approaches can provide insights into how ANKRD23 contributes to cellular energy homeostasis and potentially reveal new therapeutic targets for metabolic disorders .

What approaches can be used to investigate the role of ANKRD23 in disease models, particularly cancer?

Evidence suggests ANKRD23 may have roles in disease processes, including cancer. Researchers can investigate these connections using several antibody-based approaches:

• Expression profiling in disease tissues:

  • Compare ANKRD23 expression between normal and pathological tissues

  • Use tissue microarrays to screen multiple samples efficiently

  • Immunohistochemistry has been successfully performed on human lung cancer tissues using ANKRD23 antibodies at 1:100 dilution

• Functional studies in disease models:

  • Examine changes in proliferation, motility, and survival in cellular models with altered ANKRD23 expression

  • As demonstrated in osteosarcoma research, combine ANKRD23 antibodies with markers of cell proliferation and motility

• Signaling pathway analysis:

  • Investigate relationships with tumor suppressor p53 using anti-p53 (1:200)

  • Examine potential relationships with matrix metalloproteinases using anti-MMP2 (1:500) and anti-MMP9 (1:1000)

  • Study Akt pathway activation using anti-Akt (1:500) and anti-p-Akt2(Ser474) (1:1000)

• ANKRD23 post-translational modifications:

  • Examine how disease conditions affect ANKRD23 phosphorylation or other modifications

  • Compare modification patterns between normal and pathological states

• Genetic variant correlation:

  • Use antibodies to assess how ANKRD23 variants/mutations affect protein expression or localization

  • Correlate with disease progression or treatment response

These approaches can help elucidate ANKRD23's potential roles in disease pathogenesis and identify new biomarkers or therapeutic targets .

What are the key differences between commercially available ANKRD23 antibodies?

Several commercial ANKRD23 antibodies are available with important differences that researchers should consider:

Key considerations when selecting an antibody:

• Epitope region: Antibodies targeting different regions may have different specificities or detect different isoforms.

• Validated applications: Some antibodies are only validated for specific applications - select one that has been validated for your intended use.

• Species reactivity: Ensure the antibody recognizes ANKRD23 in your species of interest. Most available antibodies recognize human ANKRD23, while fewer are validated for mouse or other species.

• Phospho-specificity: For studying activation states, consider phospho-specific antibodies like those targeting Ser99 .

Using multiple antibodies that recognize different epitopes provides stronger validation and more confidence in experimental results .

How can I quantitatively assess ANKRD23 expression in muscle samples?

Quantitative assessment of ANKRD23 expression in muscle samples requires rigorous methodology:

• Sample preparation optimization:

  • For whole muscle: Homogenize in appropriate buffer (containing protease and phosphatase inhibitors)

  • For fiber-specific analysis: Isolate and type individual muscle fibers

  • Process samples consistently to minimize technical variation

• Western blot quantification:

  • Use stain-free technology to normalize to total protein loading

  • Include standard curves with recombinant ANKRD23 protein for absolute quantification

  • Process all samples to be compared on the same gel when possible

  • Capture images using CCD camera systems (e.g., ChemiDoc MP) for wider dynamic range

• Image analysis protocols:

  • Use software like Image Lab (version 5.2 or newer) for densitometry

  • Define consistent analysis parameters across all blots

  • Subtract local background for each band

  • Express results as relative density normalized to loading controls

• Statistical considerations:

  • Include sufficient biological replicates (minimum n=3)

  • Account for inter-individual variability in expression levels

  • Consider appropriate statistical tests based on data distribution

• Alternative quantification methods:

  • qPCR for mRNA expression correlation

  • ELISA assays for high-throughput quantification

  • Immunofluorescence with intensity quantification for spatial information

These quantitative approaches provide robust data on ANKRD23 expression levels, enabling reliable comparisons between experimental conditions .

What are the emerging research directions for ANKRD23 antibodies in biomedical research?

ANKRD23 research using antibody-based approaches is evolving in several promising directions:

• Integration of multi-omics approaches:

  • Combining antibody-based detection with transcriptomics and proteomics data

  • Correlating ANKRD23 protein expression with wider gene/protein networks

  • Establishing ANKRD23 as a potential biomarker in specific conditions

• Advanced microscopy techniques:

  • Super-resolution imaging to examine precise subcellular localization

  • Live-cell imaging with tagged antibody fragments to study dynamics

  • Expansion microscopy for enhanced spatial resolution of protein complexes

• Therapeutic potential exploration:

  • Investigation of ANKRD23 in metabolic disorders, given its role in energy metabolism

  • Examination of cancer-related roles, as suggested by osteosarcoma studies

  • Potential modulation of ANKRD23 in muscle adaptation or recovery applications

• Cross-tissue comparative studies:

  • Expanding beyond muscle to understand ANKRD23 functions in other tissues

  • Comparative analyses between cardiac, skeletal, and smooth muscle expression patterns

  • Investigation in novel disease contexts based on tissue-specific expression patterns

• Methodological advances:

  • Development of more specific antibodies against different ANKRD23 isoforms

  • Creation of phospho-specific antibodies against additional regulatory sites

  • Integration of nanobody technology for improved in vivo applications