ANKRD2 Antibody, FITC conjugated

What is ANKRD2 and why is it relevant for muscle research?

ANKRD2 (also known as Arpp) is a member of the muscle ankyrin repeat protein family, predominantly expressed in skeletal muscle where it plays a crucial role in the transcriptional response to mechanical stimulation and oxidative stress . It functions as a negative regulator of myocyte differentiation and may interact with both sarcoplasmic structural proteins and nuclear proteins to regulate gene expression during muscle development and in response to muscle stress . ANKRD2 is particularly interesting for muscle research because it participates in mechanosensory complexes in the I-band of sarcomeres and can translocate to the nucleus under stress conditions, suggesting its role as a signaling molecule .

What are the optimal fixation methods for ANKRD2 antibody staining?

For optimal ANKRD2 detection using FITC-conjugated antibodies, a 10-minute fixation with 4% paraformaldehyde at room temperature is recommended, followed by permeabilization with 0.2% Triton X-100 for 5 minutes. This preserves both sarcomeric and nuclear localization of ANKRD2. For detecting the sarcomeric form specifically, methanol fixation (-20°C for 10 minutes) may provide better results as it enhances the accessibility of structural epitopes. The fixation protocol should be validated specifically for your tissue type, as ANKRD2 expression and localization differ between slow and fast muscle fibers, with nearly double the amount in slow (type I) fibers compared to fast (type II) fibers .

What cellular compartments does ANKRD2 typically localize to?

ANKRD2 shows differential localization depending on muscle type and condition. In skeletal muscle, ANKRD2 is primarily found at the I-band of sarcomeres, where it contributes to the mechanosensory complex through interactions with titin's N2A region and calpain 3 . Under stress conditions, ANKRD2 can translocate to the nucleus, where it participates in transcriptional regulation. In cardiac muscle, both S- and M-Ankrd2 isoforms show primarily sarcomeric localization with minimal nuclear presence . FITC-conjugated ANKRD2 antibodies can effectively visualize these different localizations through appropriate immunofluorescence protocols.

How does ANKRD2 expression differ between skeletal and cardiac muscle?

ANKRD2 is expressed at significantly higher levels in skeletal muscle compared to cardiac muscle . Within skeletal muscle, ANKRD2 shows preferential expression in slow (type I) fibers compared to fast (type II) fibers, with nearly twice the amount in slow fibers from the vastus lateralis . In cardiac muscle, ANKRD2 is expressed in ventricles, the interventricular septum, and the apex of the heart, but at lower levels than in skeletal muscle . Both S- and M-Ankrd2 isoforms are present in cardiac tissue, but they show primarily sarcomeric localization rather than nuclear localization observed in stressed skeletal muscle .

How can ANKRD2 antibodies be used to investigate mechanotransduction pathways?

FITC-conjugated ANKRD2 antibodies are valuable tools for studying mechanotransduction, as they can visualize the dynamic redistribution of ANKRD2 between sarcomeres and nuclei under mechanical stress. For optimal results, researchers should combine immunofluorescence time-course studies with mechanical stretch protocols (10-15% cyclic stretch at 0.5Hz for 30 minutes) in cultured myotubes. This approach allows visualization of ANKRD2 movement from I-bands to nuclei following mechanical stimulation. The antibody can also be used to co-immunoprecipitate ANKRD2 with its binding partners like titin, calpain 3, and telethonin/TCAP to map the mechanosensory complex . This provides insights into how mechanical signals are converted to biochemical responses in muscle tissue.

What is the role of ANKRD2 in NF-κB-mediated inflammatory responses?

ANKRD2 functions as a potent repressor of inflammatory responses through direct interaction with the NF-κB pathway . FITC-conjugated ANKRD2 antibodies can be used in co-localization studies with NF-κB components (particularly p50) to visualize their interaction during inflammatory responses. Research has shown that ANKRD2 recruits p50, dominating over p65/p50 dimers, orchestrating the repression of inflammation-related genes during muscle differentiation . For studying this interaction, treat myoblasts with TNFα (10ng/ml for 30 minutes) to activate the NF-κB pathway, then perform immunofluorescence to visualize ANKRD2 and p50 co-localization. This approach helps elucidate how ANKRD2 modulates inflammatory signaling in muscle cells.

How does ANKRD2 respond to oxidative stress, and how can this be monitored?

ANKRD2 plays a significant role in oxidative stress response, and its function is regulated by Akt2-mediated phosphorylation at Ser-99 under oxidative conditions . To study this phenomenon, researchers can expose myoblasts to H₂O₂ (100-200μM for 1 hour) and then use FITC-conjugated ANKRD2 antibodies to track its subcellular redistribution. This should be complemented with phospho-specific antibodies to detect the phosphorylated form at Ser-99. Additionally, researchers can monitor the co-localization of ANKRD2 with GSK3β, as their interaction is critical for resolution of oxidative stress and inflammation via ANKRD2-dependent NF-κB inhibition .

What is the significance of ANKRD2 in muscle disease models?

ANKRD2 expression is altered in various muscle disorders, making it a valuable marker for pathological processes. In dilated cardiomyopathy patients, ANKRD2 is upregulated, with expression levels correlating with disease severity . In myoblasts from Emery-Dreifuss muscular dystrophy, ANKRD2 shows altered expression and localization patterns. FITC-conjugated ANKRD2 antibodies can be used to characterize these changes in patient-derived samples or disease models. When studying disease models, implement a quantitative immunofluorescence approach with standardized exposure settings and internal controls to accurately measure changes in ANKRD2 expression and localization relative to healthy controls.

What are the optimal protocols for ANKRD2 antibody immunofluorescence staining?

For optimal immunofluorescence results with FITC-conjugated ANKRD2 antibodies, use freshly prepared 4% paraformaldehyde for fixation (10 minutes at room temperature), followed by permeabilization with 0.2% Triton X-100 (5 minutes). Block with 5% BSA in PBS for 1 hour. The recommended dilution for primary ANKRD2 antibodies is 1:100, based on published protocols . For skeletal muscle tissue sections, a 1:1000 dilution may be more appropriate to reduce background staining. Incubate sections with the antibody at 4°C overnight in a humid chamber to prevent drying. For counterstaining, DAPI (1:1000) can be used to visualize nuclei, and phalloidin-TRITC (1:200) to visualize actin filaments, providing structural context for ANKRD2 localization.

How can researchers troubleshoot weak or non-specific ANKRD2 staining?

When encountering weak FITC-ANKRD2 antibody signals, first optimize the antibody concentration by testing a range from 1:50 to 1:200. If signal remains weak, extend the primary antibody incubation to 24-48 hours at 4°C. For high background or non-specific staining, implement these steps: (1) increase blocking time to 2 hours using 10% serum from the species in which the secondary antibody was raised; (2) include 0.1% Tween-20 in all wash buffers; (3) pre-absorb the antibody with non-specific proteins by diluting in 3% BSA with 0.1% Tween-20; (4) validate antibody specificity using ANKRD2 knockout tissues or cells as negative controls. For sarcomeric staining specifically, try methanol fixation instead of paraformaldehyde to better expose structural epitopes.

What controls should be included in ANKRD2 antibody experiments?

Rigorous controls are essential for ANKRD2 antibody experiments. Include: (1) Positive control: human skeletal muscle tissue sections or lysates, particularly from slow-twitch muscles where ANKRD2 is highly expressed ; (2) Negative control: omission of primary antibody while maintaining all other steps; (3) Specificity control: pre-incubation of antibody with recombinant ANKRD2 protein (5-10 μg/mL) before applying to samples; (4) Expression control: comparison with ANKRD2 mRNA levels by RT-qPCR; (5) Loading control: detection of housekeeping proteins (e.g., GAPDH, β-actin) when performing western blots; (6) Isotype control: use of non-specific IgG at the same concentration as the ANKRD2 antibody. These controls ensure that observed signals are specific to ANKRD2 and not artifacts.

How can photobleaching of FITC be minimized during imaging?

To minimize photobleaching of FITC-conjugated ANKRD2 antibodies during extended imaging sessions: (1) Add anti-fade agents to mounting medium (e.g., 0.1% p-phenylenediamine or commercial anti-fade solutions); (2) Reduce exposure time and illumination intensity during imaging—use the minimum settings required for adequate signal; (3) Apply oxygen scavengers such as glucose oxidase/catalase systems in the mounting medium; (4) Store slides at 4°C in the dark and image within 1-2 weeks of preparation; (5) Consider using confocal microscopy with lower light intensity at focal planes rather than wide-field microscopy; (6) For quantitative studies, capture all images in a single session with identical settings, starting with control samples to establish baseline parameters.

How should researchers quantify ANKRD2 expression and localization?

For quantitative analysis of ANKRD2 expression and localization using FITC-conjugated antibodies: (1) Capture at least 5-10 random fields per sample at identical exposure settings; (2) For expression level analysis, measure mean fluorescence intensity within defined regions of interest, normalizing to appropriate controls; (3) For subcellular localization, perform nuclear/cytoplasmic intensity ratios by co-staining with DAPI and calculating the ratio of nuclear ANKRD2 to cytoplasmic ANKRD2 signal; (4) For sarcomeric localization, use line scan analysis perpendicular to the sarcomere orientation to measure the distance between ANKRD2-positive bands relative to other sarcomeric proteins; (5) For co-localization studies, calculate Pearson's correlation coefficient or Mander's overlap coefficient between ANKRD2 and interacting proteins. Statistical analysis should compare at least three independent experiments.

What are the expected expression patterns of ANKRD2 in different muscle types?

When interpreting ANKRD2 immunofluorescence data, researchers should expect: (1) Higher expression in slow-twitch (Type I) muscle fibers compared to fast-twitch (Type II) fibers—approximately twice the amount in slow fibers from vastus lateralis ; (2) Primarily sarcomeric localization in resting muscle, with ANKRD2 concentrated at the I-band of sarcomeres; (3) In cardiac muscle, lower expression levels compared to skeletal muscle, with both S- and M-ANKRD2 isoforms showing sarcomeric localization with minimal nuclear presence ; (4) Under stress conditions, increased nuclear translocation in skeletal muscle cells, but not necessarily in cardiac muscle cells; (5) In diseased tissue like dilated cardiomyopathy, significantly elevated expression correlating with disease severity .

How can ANKRD2 antibodies be used in multi-parameter flow cytometry?

For multi-parameter flow cytometry applications with FITC-conjugated ANKRD2 antibodies: (1) Optimize cell permeabilization (0.1% saponin or 0.3% Triton X-100) to access intracellular ANKRD2 while preserving cellular integrity; (2) Use fluorophore combinations that minimize spectral overlap with FITC (e.g., PE-Cy7 for additional markers); (3) Implement hierarchical gating strategies—first identify myoblasts/myocytes using markers like desmin or MyoD, then analyze ANKRD2 expression within this population; (4) For quantification, report median fluorescence intensity rather than mean values, as the former is less affected by outliers; (5) Use compensation controls and fluorescence-minus-one (FMO) controls to accurately set gates. This approach enables quantitative analysis of ANKRD2 expression across different cell populations and experimental conditions.

What emerging techniques could enhance ANKRD2 research?

Emerging techniques that could revolutionize ANKRD2 research include: (1) Super-resolution microscopy (STORM, PALM, or SIM) to visualize ANKRD2's precise localization within sarcomeric structures at nanometer resolution; (2) Live-cell imaging using ANKRD2-GFP fusion proteins combined with FITC-antibody pulse-chase experiments to track dynamic movement between cellular compartments; (3) Proximity ligation assays to visualize and quantify ANKRD2 interactions with binding partners like p50, titin, or calpain 3 in situ; (4) CRISPR-Cas9 genome editing to create tagged endogenous ANKRD2, allowing physiological expression level monitoring; (5) Single-cell RNA-seq combined with ANKRD2 protein detection to correlate transcriptional programs with protein expression at the single-cell level in heterogeneous muscle tissues.