ANKRD2 Antibody, Biotin conjugated

What is ANKRD2 and what is its biological significance?

ANKRD2 (ankyrin repeat domain 2) is a stretch-responsive muscle protein that functions as a negative regulator of myocyte differentiation. This protein plays a critical role in mechanotransduction, participating in the mechanical stress response primarily in muscle fibers. ANKRD2 interacts with both sarcoplasmic structural proteins and nuclear proteins to regulate gene expression during muscle development and in response to muscle stress . The protein has a calculated molecular weight of 37 kDa, though it is typically observed at approximately 42 kDa in experimental conditions .

ANKRD2 has been identified in various tissues beyond muscle, including cell lines derived from human osteosarcoma, suggesting broader biological functions than initially understood . Its involvement in stress response pathways makes it an important target for studies investigating mechanical adaptation in tissues and disease pathogenesis.

What applications are ANKRD2 antibodies suitable for in research?

ANKRD2 antibodies have been validated for multiple experimental applications, with documented effectiveness in various techniques:

For optimal results, each antibody should be titrated in the specific testing system being used . When working with biotin-conjugated variants, researchers should consider the potential for increased sensitivity in detection systems that utilize streptavidin complexes while also accounting for possible background from endogenous biotin.

How should researchers optimize storage conditions for ANKRD2 antibodies to maintain reactivity?

ANKRD2 antibodies require specific storage conditions to maintain their reactivity and specificity. Standard unconjugated antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 and should be stored at -20°C, where they remain stable for approximately one year after shipment . For small volume preparations (around 20μl), manufacturers often include 0.1% BSA to improve stability .

For biotin-conjugated antibodies, additional precautions should be taken as the biotin-streptavidin interaction can be affected by repeated freeze-thaw cycles. It is recommended to:

• Aliquot the antibody upon receipt to minimize freeze-thaw cycles

• Avoid exposure to direct light, which can affect biotin activity

• Maintain sterile conditions to prevent microbial contamination

• Monitor for signs of precipitation or aggregation, which may indicate reduced functionality

When working with antibodies in experimental settings, researchers should prepare working dilutions just before use rather than storing diluted solutions for extended periods.

What considerations are important when selecting between monoclonal and polyclonal ANKRD2 antibodies?

The choice between monoclonal and polyclonal ANKRD2 antibodies should be guided by experimental goals and sample characteristics:

Polyclonal antibodies (such as the rabbit polyclonal 11821-1-AP ):

• Recognize multiple epitopes on the target protein

• Often provide robust signals in applications like Western blotting

• May show broader reactivity across species (human, mouse, rat)

• Can compensate for protein modifications or conformational changes

• Potentially higher risk of cross-reactivity with similar proteins

Monoclonal antibodies (such as the rabbit recombinant monoclonal EPR10731(B) ):

• Target a single epitope with high specificity

• Provide consistent lot-to-lot reproducibility

• Often preferred for quantitative applications

• May be more sensitive to epitope masking or denaturation

• Typically show more restricted species reactivity

For biotin-conjugated variants, the conjugation process may differentially affect epitope recognition between monoclonal and polyclonal antibodies, making validation in the specific experimental context crucial.

What methodological approaches can optimize the detection of ANKRD2 in tissue sections using biotin-conjugated antibodies?

Detection of ANKRD2 in tissue sections requires careful methodological consideration when using biotin-conjugated antibodies. Research has shown that enzymatic biotinylation provides optimal specificity and sensitivity for detecting protein targets in paraffin-embedded tissues . When working with ANKRD2 antibodies, consider the following protocol optimizations:

• Antigen retrieval optimization: For ANKRD2 detection, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 can serve as an alternative . The effectiveness of retrieval methods should be empirically determined for each tissue type.

• Signal amplification considerations: Direct detection of biotin with streptavidin-coupled HRP using 3,3'-diaminobenzidine as a substrate can yield specific membranous staining patterns without background interference . In contrast, conventional signal amplification with anti-biotin antibodies followed by HRP-polymer-coupled secondary antibodies may introduce background staining .

• Titration and concentration optimization: Even at low concentrations, high-affinity binding molecules can saturate available epitopes. For example, designed ankyrin repeat proteins with picomolar affinity demonstrated that concentrations as low as 1 μg/ml provided adequate coverage without generating non-specific binding . Similar principles may apply to high-affinity biotin-conjugated ANKRD2 antibodies.

• Blocking endogenous biotin: In tissues with high endogenous biotin (like kidney, liver, and brain), pretreatment with avidin/biotin blocking reagents is essential to minimize background and false positive signals.

How can ANKRD2 antibodies be utilized to investigate the role of ANKRD2 in disease progression and therapeutic response?

ANKRD2 has emerged as a potential biomarker in several pathological contexts, and antibody-based detection methods offer valuable insights into disease mechanisms. A longitudinal serum biomarker study identified ANKRD2 as one of the proteins differentially expressed in non-ambulant patients with muscle-related disorders, with expression levels significantly affected by treatment with glucocorticosteroids (p = 0.0005) .

For researchers investigating disease progression:

• Longitudinal sample analysis: ANKRD2 antibodies can be used to track protein expression changes over time in patient cohorts. Analysis should incorporate appropriate statistical methods such as linear mixed models to account for repeated measurements .

• Integration with clinical parameters: When analyzing ANKRD2 expression, researchers should correct for clinical variables that may affect results. Studies have shown significant differences in protein expression profiles across clinical centers, highlighting the importance of standardized protocols and statistical corrections .

• Multiplexed detection approaches: ANKRD2 can be analyzed alongside other biomarkers for comprehensive profiling. For example, one study identified 21 proteins whose levels significantly decrease with age, including MDH2, ETFA, MYL3, NES, CK, CA3, MYOM3, LDHB, COL1A1, ENO3, BASP1, TNNT3, MAP4, TTN, DES, TNNT2, AKAP1, ANKRD2, HDAC2, LCP1, and KRT10 . Multiplexed assays using differentially labeled antibodies can provide insights into pathway interactions.

• Treatment response monitoring: Biotin-conjugated ANKRD2 antibodies can be particularly valuable for assessing therapeutic responses due to their enhanced sensitivity in detection systems. Studies have demonstrated that treatment with corticosteroids partly counteracts the effect of disease progression on ANKRD2 expression levels .

What are the considerations for using ANKRD2 antibodies in studying the protein's role in mechanotransduction pathways?

ANKRD2 functions as a mechano-sensor protein primarily expressed in muscle fibers, where it participates in the mechanical stress response . When designing experiments to investigate ANKRD2's role in mechanotransduction:

• Selection of appropriate experimental models: Since ANKRD2 responds to mechanical stress, in vitro models should incorporate relevant mechanical stimulation. For skeletal muscle studies, C2C12 myoblasts subjected to cyclic stretch can be used, while for cardiac muscle, primary cardiomyocytes or H9c2 cells with pressure or stretch challenges are appropriate.

• Co-localization studies: Biotin-conjugated ANKRD2 antibodies can be utilized in combination with other fluorophore-conjugated antibodies for co-localization studies to identify interaction partners during mechanical stress. This approach can help elucidate how ANKRD2 translocates between sarcomeric structures and nuclei under different mechanical conditions.

• Subcellular fractionation validation: When studying ANKRD2 translocation between cellular compartments, researchers should validate subcellular fractionation using appropriate markers for sarcomeric structures, nuclear compartments, and other relevant organelles.

• Experimental validation through genetic manipulation: Results obtained with ANKRD2 antibodies should be validated using genetic approaches such as knockdown or overexpression systems. Recent studies examining ectopic expression of ANKRD2 in osteosarcoma cells demonstrated effects on proliferation, motility, and other cancer-related processes .

How do designed ankyrin repeat proteins (DARPins) compare to conventional ANKRD2 antibodies for research applications?

Designed ankyrin repeat proteins (DARPins) represent an emerging alternative to traditional antibodies, with distinct advantages and limitations for detecting target proteins:

Research has demonstrated that DARPins with picomolar binding affinity (KD ~90 pM) can achieve specific and sensitive detection of target proteins in paraffin-embedded tissue sections . For optimal results with DARPins, direct detection of biotin on genetically fused AviTags with streptavidin-coupled HRP has proven more specific than conventional signal amplification with anti-biotin antibodies .

When considering DARPins as alternatives to ANKRD2 antibodies, researchers should note that the affinity of the DARPin is crucial, but making a picomolar binder multivalent does not necessarily provide additional benefits in terms of detection sensitivity . This suggests that optimizing the detection method may be more important than increasing binding valency.

What protocols should be considered when using ANKRD2 antibodies in the investigation of protein-protein interactions?

Investigating ANKRD2's interactions with other proteins requires careful selection of methodologies and antibody applications:

• Immunoprecipitation optimization: For IP applications, ANKRD2 antibodies should be used at concentrations of 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate . When using biotin-conjugated antibodies for IP, researchers must carefully design protocols to avoid interference from the biotin-streptavidin system during subsequent detection steps.

• Cross-linking strategies: Since ANKRD2 interacts with both sarcoplasmic structural proteins and nuclear proteins , transient interactions may require chemical cross-linking prior to immunoprecipitation. Common cross-linkers like DSP (dithiobis(succinimidyl propionate)) or formaldehyde can stabilize complexes before cell lysis.

• Proximity ligation assays: Biotin-conjugated ANKRD2 antibodies can be paired with antibodies against putative interaction partners in proximity ligation assays (PLA) to visualize protein-protein interactions in situ with high sensitivity and specificity.

• Co-immunoprecipitation validation: When identifying novel interaction partners, researchers should validate findings using reciprocal co-immunoprecipitation and alternative methods such as pull-down assays with recombinant proteins.

• Controls for specificity: Include appropriate negative controls using non-specific antibodies of the same isotype, and positive controls targeting known ANKRD2 interaction partners. For biotin-conjugated antibodies, additional controls should address potential streptavidin binding to endogenous biotinylated proteins.

What factors affect the observed molecular weight of ANKRD2 in experimental systems?

Researchers often observe discrepancies between the calculated and experimental molecular weights of ANKRD2. While the calculated molecular weight is reported as 37 kDa (2 kDa for some fragments), the observed molecular weight in Western blot applications is typically around 42 kDa . Several factors can contribute to this discrepancy:

• Post-translational modifications: ANKRD2 may undergo phosphorylation, glycosylation, or other modifications that alter its electrophoretic mobility.

• Protein structure and SDS binding: The ankyrin repeat domains may bind SDS differently than globular proteins used as molecular weight standards.

• Isoform variation: Different splice variants or isoforms of ANKRD2 may be present in different tissues or experimental systems.

• Sample preparation conditions: Denaturation temperature, reducing agent concentration, and buffer composition can all affect the apparent molecular weight.

When interpreting Western blot results, researchers should consider using positive controls from tissues known to express ANKRD2, such as skeletal muscle tissue from human or mouse sources , to establish appropriate size references.

How can cross-reactivity issues be addressed when working with ANKRD2 antibodies?

Cross-reactivity can complicate the interpretation of results when using ANKRD2 antibodies. To minimize and address potential cross-reactivity:

• Validation in multiple systems: Confirm antibody specificity using multiple techniques. For ANKRD2 antibodies, positive detection has been demonstrated in Western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence across various sample types .

• Knockout/knockdown controls: Utilize ANKRD2 knockout or knockdown systems as negative controls. Published applications have demonstrated the utility of this approach for validating antibody specificity .

• Epitope mapping: Understanding the specific epitope recognized by an antibody can help predict potential cross-reactivity with structurally similar proteins. For polyclonal antibodies raised against ANKRD2 fusion proteins, consider the sequence homology of the immunogen with other ankyrin repeat-containing proteins.

• Multiple antibody approach: Use multiple antibodies targeting different epitopes of ANKRD2 to confirm results. Consistent findings across different antibodies provide stronger evidence for specificity.

• Pre-absorption controls: For critical applications, pre-absorb the antibody with recombinant ANKRD2 protein to confirm that the observed signal is specifically competed away.

What are the optimal dilution strategies for biotin-conjugated ANKRD2 antibodies across different applications?

The optimal dilution of biotin-conjugated ANKRD2 antibodies varies by application and must be empirically determined. Based on recommended dilutions for unconjugated ANKRD2 antibodies , the following starting points can be considered:

For biotin-conjugated antibodies specifically, consider these additional factors:

• The degree of biotinylation affects optimal dilution (more biotin may require higher dilution)

• Detection system sensitivity (HRP-streptavidin vs. fluorescent-streptavidin)

• Endogenous biotin levels in the sample

Titration experiments should systematically compare dilutions to identify the concentration that provides maximum specific signal with minimal background. It is recommended that antibody reagents should be carefully titrated in each testing system to obtain optimal results .

How can ANKRD2 antibodies contribute to understanding the protein's role in cancer progression?

Recent research has revealed unexpected roles for ANKRD2 in cancer biology, particularly in osteosarcoma. While ANKRD2 is primarily known as a muscle-specific protein, studies have demonstrated its expression in osteosarcoma cell lines and its involvement in the pathogenesis of this disease . Researchers investigating ANKRD2's role in cancer should consider:

• Expression profiling across cancer types: Biotin-conjugated ANKRD2 antibodies can be used in tissue microarrays to assess expression patterns across different cancer types and stages, potentially revealing correlations with clinical outcomes.

• Functional studies in cancer models: Research has shown that ectopic expression of ANKRD2 affects proliferation, motility, and other cancer-related processes in osteosarcoma models . Similar approaches can be applied to other cancer types where mechano-sensing pathways may be dysregulated.

• Mechanism investigation: ANKRD2 may function as a "double-faced" cancer driver gene, with context-dependent roles in cancer progression . Antibody-based techniques can help elucidate how ANKRD2 interacts with cancer-related signaling pathways.

• Translational research applications: If ANKRD2 expression correlates with clinical outcomes or treatment responses, antibody-based detection methods could potentially be developed into diagnostic or prognostic tools.

What considerations are important when using ANKRD2 antibodies in multiplexed detection systems?

Multiplexed detection allows simultaneous analysis of multiple proteins, providing insights into pathway interactions and regulatory networks. When incorporating ANKRD2 antibodies into multiplexed systems:

• Panel design: Consider protein co-expression patterns when designing multiplexed panels. ANKRD2 has been identified alongside other biomarkers such as MDH2, ETFA, MYL3, and others that show significant changes during aging or disease progression .

• Technical compatibility: Ensure that detection methods for biotin-conjugated ANKRD2 antibodies are compatible with other detection systems in the multiplex. If using streptavidin-based detection, other antibodies should utilize different detection chemistry.

• Signal separation: In multiplexed fluorescence applications, select fluorophores with minimal spectral overlap and include appropriate controls for spillover compensation.

• Antibody cross-reactivity: Test antibodies individually before combining them in multiplexed assays to ensure each antibody maintains specificity when used in combination.

• Data analysis approaches: Complex multiplexed datasets require appropriate statistical methods. Studies investigating ANKRD2 alongside other biomarkers have successfully employed methods such as F-tests to identify differentially expressed proteins across treatment groups, with Benjamini-Hochberg procedures to correct for multiple testing .

How can researchers validate experimental findings related to ANKRD2 functional studies?

Validation of ANKRD2 functional studies requires a multi-faceted approach:

• Genetic manipulation validation: When investigating ANKRD2 function through overexpression or knockdown, validate expression changes at both mRNA (qPCR) and protein (Western blot) levels using validated ANKRD2 antibodies .

• Rescue experiments: For knockdown studies, perform rescue experiments with wild-type or mutant ANKRD2 to confirm specificity of observed phenotypes.

• Orthogonal detection methods: Confirm ANKRD2 expression patterns using different types of antibodies (monoclonal vs. polyclonal) or alternative detection methodologies like mass spectrometry.

• Biological replicates and statistical analysis: Ensure adequate biological replication and appropriate statistical analysis. Studies examining ANKRD2 as a biomarker have employed sophisticated statistical approaches such as linear mixed models for longitudinal data analysis .

• Cross-species validation: ANKRD2 antibodies have demonstrated reactivity across human, mouse, and rat samples , enabling validation of findings across species models where appropriate.

• In vitro to in vivo translation: When possible, validate in vitro findings in appropriate animal models using ANKRD2 antibodies that have been validated for in vivo applications.