ANKRD2 Antibody, HRP conjugated

What are the primary applications for ANKRD2 antibody with HRP conjugation?

ANKRD2 antibody with HRP conjugation is primarily designed for Western Blot (WB) applications, offering direct detection without the need for secondary antibodies . The unconjugated form of ANKRD2 antibody has broader applications including Western Blot (WB), Immunoprecipitation (IP), Immunohistochemistry (IHC), and Immunofluorescence (IF)/Immunocytochemistry (ICC) . When selecting the HRP-conjugated variant, researchers should consider their specific detection requirements and whether direct detection is advantageous for their experimental design.

What is the recommended working dilution for ANKRD2 antibody?

The optimal working dilution depends on the specific application:

It is strongly recommended to titrate the antibody in each testing system to obtain optimal results, as the performance may be sample-dependent . For HRP-conjugated ANKRD2 antibodies, similar principles apply, though specific optimization might be required due to the direct detection format.

What is the molecular weight of ANKRD2 protein?

While the calculated molecular weight of ANKRD2 is reported as 37 aa, 2 kDa, the observed molecular weight in protein detection systems is typically around 42 kDa . This discrepancy between calculated and observed molecular weights is common for many proteins and could be attributed to post-translational modifications or the protein's structural properties. When analyzing Western blot results, researchers should expect to observe a band around 42 kDa.

How should I optimize antigen retrieval for ANKRD2 immunohistochemistry?

For optimal antigen retrieval in IHC applications with ANKRD2 antibody, it is recommended to use TE buffer at pH 9.0 . Alternatively, citrate buffer at pH 6.0 can be employed if TE buffer does not yield satisfactory results. The choice between these methods may depend on your specific tissue type and fixation protocol. When working with highly fixated tissues or challenging samples, extended retrieval times may be necessary. Always include positive control tissues (such as human skeletal muscle) to validate the retrieval protocol, as ANKRD2 is known to be highly expressed in skeletal muscle tissue.

What controls should I include when using ANKRD2 antibody in silencing studies?

When designing experiments involving ANKRD2 silencing, three critical controls should be included to ensure valid interpretation:

• Non-silenced cells infected with control vectors (e.g., AAV-shLuc)

• Uninfected control cells

• Validation of silencing efficiency at both RNA and protein levels

This approach was successfully employed in studies examining the effects of Ankrd2 silencing in human skeletal muscle cells, where researchers verified significant reduction in Ankrd2 expression compared to both control conditions . Additionally, inclusion of isotype controls and secondary antibody-only controls will help distinguish specific from non-specific binding when using the antibody to validate knockdown efficiency.

How can I validate the specificity of ANKRD2 antibody detection?

To validate ANKRD2 antibody specificity, a multi-faceted approach is recommended:

• Perform experiments with blocking peptides such as the specific blocking peptide available for anti-ANKRD2 (ARP42559_P050-HRP) antibody (Catalog # AAP42559)

• Include positive control tissues known to express ANKRD2 (skeletal muscle tissue is strongly recommended)

• Include negative controls where ANKRD2 is known to be absent or has been knocked down using validated siRNA/shRNA constructs

• Compare results with alternative ANKRD2 antibodies targeting different epitopes

• For definitive validation, utilize tissues or cells from ANKRD2 knockout models when available

This comprehensive approach ensures that the signal detected truly represents ANKRD2 protein rather than cross-reactive epitopes.

Why might I observe multiple bands when using ANKRD2 antibody in Western blot?

The detection of multiple bands in Western blot using ANKRD2 antibody may occur for several reasons:

• ANKRD2 has been reported to exist in different isoforms or splice variants

• Post-translational modifications such as phosphorylation, as evidenced by studies developing phospho-specific antibodies against ANKRD2 at Ser99

• Proteolytic degradation of the protein during sample preparation

• Cross-reactivity with related proteins, especially other MARP family members (ANKRD1/CARP and DARP)

To address this issue, optimize your sample preparation by including appropriate protease inhibitors, reducing sample heating time, and ensuring thorough blocking. If additional bands persist, consider performing peptide competition assays with the immunizing peptide or using samples with ANKRD2 knockdown to identify the specific band representing ANKRD2.

What are common issues with storage and handling of HRP-conjugated ANKRD2 antibodies?

HRP-conjugated antibodies require special handling considerations to maintain optimal activity:

• Store in light-protected vials or cover with light-protecting material (e.g., aluminum foil) to prevent photobleaching

• HRP-conjugated antibodies are typically stable for at least 12 months at 4°C

• For extended storage (up to 24 months), dilute with up to 50% glycerol and store at -20°C to -80°C

• Avoid repeated freeze-thaw cycles as this will compromise both enzyme activity and antibody binding

• Allow the antibody to reach room temperature before opening to prevent condensation

When handling issues occur, reduced signal intensity is the most common symptom, which may be misinterpreted as low protein expression rather than antibody degradation.

How can I optimize detection in tissues with low ANKRD2 expression?

For tissues with low ANKRD2 expression, consider these optimization strategies:

• Use more concentrated antibody dilutions within the recommended range

• Extend primary antibody incubation time (overnight at 4°C may improve signal)

• Employ signal amplification systems compatible with HRP detection

• For IHC applications, optimize antigen retrieval conditions thoroughly

• Consider using more sensitive detection substrates for HRP

• Increase protein loading for Western blot applications, while ensuring equal loading with appropriate controls

Remember that ANKRD2 expression is tissue-specific, with highest expression in skeletal muscle, so validation in these tissues should precede attempts to detect low expression in other tissue types.

How does ANKRD2 function in mechanosensing pathways?

ANKRD2 (also known as Arpp) belongs to the MARP (Muscle Ankyrin Repeat Protein) family of mechanosensing proteins, forming a complex with titin (N2A), calpain 3 protease, and myopalladin . In muscle tissue, ANKRD2 is primarily located in the I-band of the sarcomere and translocates to the nucleus of adjacent myofibers following muscle injury, suggesting a role in the mechanical stress response .

Research methodologies to study ANKRD2's mechanosensing function include:

• Mechanical stretch experiments in cultured myocytes with subsequent analysis of ANKRD2 localization

• Co-immunoprecipitation studies to identify interaction partners under various mechanical stress conditions

• Expression profiling in models of muscle injury or mechanical loading

• ANKRD2 silencing experiments to elucidate downstream effects on mechanosensitive pathways

Understanding ANKRD2's mechanosensing role is critical for research in muscle physiology, adaptation to exercise, and muscular dystrophies.

What signaling pathways are affected by ANKRD2 modulation?

Expression profiling of ANKRD2-silenced human myotubes has revealed that ANKRD2 affects multiple signaling pathways, including:

• Intercellular communication pathways:

  • Cytokine-cytokine receptor interaction

  • Endocytosis

  • Focal adhesion

  • Tight junction

  • Gap junction

  • Regulation of actin cytoskeleton

• Intracellular signaling pathways:

  • Calcium signaling

  • Insulin signaling

  • MAPK signaling

  • p53 signaling

  • TGF-β signaling

  • Wnt signaling

Methodologically, researchers investigating these pathways should consider using phospho-specific antibodies to track signaling cascade activation, combined with ANKRD2 manipulation through overexpression or silencing approaches. The broad impact of ANKRD2 on these pathways suggests its role as a nodal point in integrating mechanical stimuli with cellular adaptation responses.

What is the significance of ANKRD2 in cancer research?

Recent studies have investigated ANKRD2's potential role in cancer progression, particularly in osteosarcoma. Research has shown that ectopic expression of Ankrd2 can affect proliferation and motility of cancer cells . When designing studies to investigate ANKRD2 in cancer contexts, researchers should:

• Compare ANKRD2 expression between normal and cancerous tissues of the same origin

• Analyze correlation between ANKRD2 expression and clinical outcomes

• Perform functional studies with ANKRD2 overexpression or knockdown to assess effects on:

  • Cell proliferation

  • Migration and invasion

  • Resistance to apoptosis

  • Response to therapeutic agents

The interaction of ANKRD2 with p53, a major tumor suppressor, further supports its potential significance in cancer biology, as p53 has been identified as an upstream effector of the ANKRD2 gene .

How do ANKRD2 interactions with PDZ and SH3 domain proteins contribute to its function?

ANKRD2 has been shown to interact with proteins containing PDZ and SH3 domains, which are known to be involved in signaling pathways . These interactions support ANKRD2's role in mediating signals between different cellular compartments, particularly from the nucleus to the cytoskeleton.

To study these interactions, researchers can employ:

• Yeast two-hybrid screening to identify novel interaction partners

• Co-immunoprecipitation followed by mass spectrometry

• Proximity ligation assays to confirm interactions in situ

• Truncation and mutation studies to identify specific binding domains

The interaction with PDZ-Lim protein family members is particularly significant, as these proteins mediate signals from the nucleus to the cytoskeleton, aligning with ANKRD2's proposed role in mechanotransduction .

How can I design experiments to investigate ANKRD2's transcriptional regulation?

ANKRD2 has been shown to be regulated by several transcription factors, including Nkx2.5, p53, and can be modulated by Ankrd1/CARP . To investigate ANKRD2's transcriptional regulation:

• Perform reporter gene assays using the ANKRD2 promoter

• Conduct chromatin immunoprecipitation (ChIP) assays to identify transcription factor binding sites

• Use EMSA (Electrophoretic Mobility Shift Assay) to confirm direct binding

• Employ site-directed mutagenesis of potential binding sites to validate their functionality

• Analyze ANKRD2 expression in response to overexpression or knockdown of suspected transcriptional regulators

Interestingly, research has identified a potential regulatory feedback loop where transcription factors PAX6, LHX2, NFIL3, and MECP2 can bind both the Ankrd2 protein and its promoter , suggesting complex autoregulatory mechanisms.

What are the best approaches for studying ANKRD2 subcellular localization?

ANKRD2 exhibits dynamic subcellular localization, being predominantly nuclear in myoblasts and shifting to the cytoplasm upon differentiation, and moving to the nucleus of adjacent myofibers following muscle injury . To study this dynamic localization:

• Use immunofluorescence with the ANKRD2 antibody (dilution 1:50-1:500) in various experimental conditions

• Combine with counterstaining for nuclear (DAPI) and sarcomeric markers

• Perform subcellular fractionation followed by Western blot analysis

• Create GFP-tagged ANKRD2 constructs for live-cell imaging

• Induce mechanical stress or injury to observe translocation events

Control experiments should include validation of fractionation purity using compartment-specific markers, and comparison of endogenous versus tagged protein localization patterns to ensure tag-related artifacts are not present.