ARMC10 Antibody

What is ARMC10 and why is it significant in research?

ARMC10 (armadillo repeat containing 10) is a protein that contains an armadillo repeat and transmembrane domain. It has emerged as a significant research target due to its dual localization in mitochondria and cell nuclei, and its crucial role in regulating mitochondrial dynamics and trafficking . ARMC10 has been implicated in neuroprotection, axon regeneration, and cellular response to stress conditions . Its significance extends to potential therapeutic applications in neurodegenerative diseases, ischemic stroke, and axonal injuries .

What are the characteristics of commercially available ARMC10 antibodies?

ARMC10 antibodies are primarily available as rabbit polyclonal antibodies that detect endogenous levels of total ARMC10 . These antibodies recognize multiple ARMC10 isoforms, typically detecting bands between 31-37 kDa in Western blots, despite the calculated molecular weight of 38 kDa . Current commercially available antibodies show reactivity with human, mouse, and rat samples and have been validated for multiple applications including Western blot, immunohistochemistry, immunofluorescence, and immunoprecipitation .

How should I optimize Western blot protocols for ARMC10 detection?

For optimal ARMC10 detection via Western blot, follow these methodological considerations:

• Sample preparation: Use complete lysis buffers containing protease inhibitors to preserve all ARMC10 isoforms. For mitochondrial enrichment studies, perform subcellular fractionation before Western blotting .

• Gel percentage: Use 10-12% SDS-PAGE gels for optimal separation of the 31-37 kDa ARMC10 isoforms .

• Primary antibody incubation: Dilute ARMC10 antibody between 1:500-1:3000 in blocking solution and incubate overnight at 4°C for best results .

• Multiple band interpretation: Be aware that ARMC10 antibodies may detect up to four bands in Western blots, corresponding to different isoforms. The most prominent band in brain tissue appears at approximately 31 kDa, likely corresponding to the C and/or E isoforms .

• Controls: Include positive controls such as A431 cells, HEK-293 cells, or HepG2 cells, which have been validated to express detectable levels of ARMC10 .

What are the best practices for immunofluorescence experiments using ARMC10 antibodies?

For successful immunofluorescence experiments with ARMC10 antibodies:

• Fixation method: Use 4% paraformaldehyde for 10-15 minutes at room temperature to preserve both mitochondrial morphology and ARMC10 localization .

• Permeabilization: Use 0.1-0.2% Triton X-100 for 10 minutes to allow antibody access while maintaining subcellular structures .

• Antibody dilution: Use ARMC10 antibodies at 1:50-1:500 dilution, with higher concentrations recommended for neuronal cultures .

• Co-staining strategies: For mitochondrial localization studies, co-stain with mitochondrial markers such as MitoTracker or antibodies against mitochondrial proteins (e.g., TOMM20) .

• Nuclear versus mitochondrial signal interpretation: Be aware that ARMC10 shows bimodal localization, with signals in both nuclei (typically stronger) and mitochondria (typically fainter), particularly in neurons .

How can I validate the specificity of ARMC10 antibodies in my experimental system?

To ensure antibody specificity:

• Knockdown validation: Perform siRNA or shRNA-mediated knockdown of ARMC10 and confirm reduction of signal in Western blot or immunostaining. Research has validated shRNA sequences targeting the C-terminal region of ARMC10 to knockdown all endogenous isoforms .

• Overexpression controls: Use ARMC10-GFP or other tagged constructs as positive controls, comparing endogenous and overexpressed signal patterns .

• Peptide competition: Pre-incubate the antibody with immunizing peptide to block specific binding, which should eliminate true ARMC10 signals.

• Cross-species validation: Verify antibody reactivity in multiple species (human, mouse, rat) if working with animal models .

• Multiple antibody validation: When possible, compare results using antibodies from different vendors or those targeting different epitopes of ARMC10 .

How can ARMC10 antibodies be used to study mitochondrial dynamics?

ARMC10 antibodies are valuable tools for investigating mitochondrial dynamics through these advanced approaches:

• Mitochondrial morphology analysis: Use immunofluorescence with ARMC10 antibodies to quantify mitochondrial size, shape, and distribution. Research has shown that ARMC10 overexpression affects mitochondrial aggregation in HEK293T cells and mildly affects aggregation in neurons .

• Live-cell imaging: Combine ARMC10 antibody immunostaining with time-lapse microscopy to correlate ARMC10 expression levels with dynamic changes in mitochondrial morphology.

• Co-immunoprecipitation: Use ARMC10 antibodies to pull down protein complexes involved in mitochondrial dynamics. Studies have demonstrated that ARMC10 interacts with the KIF5/Miro1-2/Trak2 trafficking complex .

• Phosphorylation studies: Use phospho-specific antibodies alongside total ARMC10 antibodies to study how AMPK-mediated phosphorylation at S45 affects ARMC10 function in mitochondrial dynamics .

• Stress-induced mitochondrial fragmentation: Examine how ARMC10 expression levels affect mitochondrial fragmentation under stress conditions, particularly in Aβ-induced toxicity models where ARMC10 overexpression has been shown to prevent mitochondrial fission .

What is the role of ARMC10 in neuroprotection and how can antibodies help elucidate this function?

ARMC10 antibodies can help investigate neuroprotective mechanisms through:

• Cellular stress models: Use ARMC10 antibodies to track protein expression and localization changes during oxidative stress, Aβ exposure, or ischemia/reperfusion injury. Research has shown that ARMC10 overexpression protects against Aβ-induced mitochondrial fragmentation and neuronal death .

• Signaling pathway analysis: Combine ARMC10 antibodies with antibodies against Wnt/β-catenin pathway components (β-catenin, GSK-3β, p-GSK-3β) to elucidate how ARMC10 activates this pathway for neuroprotection .

• Apoptotic marker correlation: Perform dual immunostaining with ARMC10 antibodies and apoptotic markers (Bcl-2, Bax) to establish correlations between ARMC10 expression and neuronal survival. Studies have demonstrated that ARMC10 expression affects the levels of these apoptotic markers .

• Mitochondrial functional assays: Combine ARMC10 immunostaining with functional assays (ROS production, ATP levels) to connect ARMC10 expression to mitochondrial function in neuronal health .

• In vivo stroke models: Use ARMC10 antibodies in tissue sections from ischemic stroke models to track expression changes during injury and recovery phases .

How should I interpret contradictory ARMC10 localization data in different cell types?

When facing contradictory localization data:

• Cell-type specific expression patterns: ARMC10 shows differential localization patterns between cell types. While predominantly mitochondrial in HEK293T cells, it shows stronger nuclear staining in neuronal populations .

• Isoform-specific localization: The four ARMC10 isoforms may localize differently. Use isoform-specific antibodies or tagged constructs of specific isoforms to differentiate their localization patterns .

• Physiological state considerations: ARMC10 localization may change with cellular stress, differentiation state, or cell cycle phase. Document experimental conditions carefully when comparing results.

• Technical considerations: Fixation and permeabilization methods significantly affect observed localization patterns. Compare protocols when resolving contradictory results. In particular, mitochondrial staining can be more sensitive to technical variations than nuclear staining .

• Quantitative analysis: When possible, perform quantitative analysis of subcellular fractionation studies with Western blot to complement immunofluorescence data for more accurate localization assessment .

How can ARMC10 antibodies be applied in ischemic stroke research?

ARMC10 antibodies have specific applications in ischemic stroke research:

• Expression profiling: Clinical studies have shown that ARMC10 expression is significantly decreased in ischemic stroke patients compared to controls. Use ARMC10 antibodies for immunoblotting to quantify expression changes in patient samples or experimental models .

• OGD/R cellular models: In oxygen-glucose deprivation/reperfusion (OGD/R) models of ischemic stroke, ARMC10 antibodies can track expression changes during the injury and recovery phases .

• Therapeutic intervention assessment: Use ARMC10 antibodies to evaluate how potential neuroprotective interventions affect ARMC10 expression and localization after ischemic injury.

• Wnt/β-catenin pathway activation: Combine ARMC10 immunostaining with β-catenin and GSK-3β detection to study how ARMC10 activates this pathway during ischemic injury. Research has shown that ARMC10 affects mitochondrial function and neuronal apoptosis through the Wnt/β-catenin signaling pathway in OGD/R models .

• Mitochondrial morphology assessment: Use ARMC10 antibodies alongside mitochondrial markers to track how ischemia-reperfusion affects mitochondrial dynamics and how this correlates with ARMC10 expression levels .

What is the relationship between ARMC10 and axon regeneration, and how can antibodies investigate this connection?

ARMC10 antibodies can help explore the role in axon regeneration through:

• Receptor identification: Research has identified ARMC10 (ArmC10) as a high-affinity receptor for oncomodulin (Ocm), which promotes axon regeneration. Use co-immunoprecipitation with ARMC10 antibodies to study this interaction in different neuronal populations .

• Optic nerve injury models: Use ARMC10 antibodies to study expression in retinal ganglion cells after optic nerve injury, where ARMC10 has been shown to be necessary for inflammation- and Ocm-mediated regeneration .

• Peripheral nerve regeneration: Apply ARMC10 antibodies to investigate expression changes during peripheral nerve regeneration, which ARMC10 has been shown to accelerate .

• Spinal cord injury models: Use immunohistochemistry with ARMC10 antibodies to track expression in dorsal root ganglion neurons after spinal cord injury, where ARMC10 promotes axon regeneration .

• Human iPSC-derived sensory neurons: Apply ARMC10 antibodies to study expression in human induced pluripotent stem cell-derived sensory neurons, where Ocm has been shown to promote neurite outgrowth through ARMC10 .

How can I troubleshoot weak or absent ARMC10 signal in Western blot experiments?

When facing detection issues:

• Sample preparation optimization: Ensure complete lysis using buffers containing 1% Triton X-100 or RIPA buffer with protease inhibitors. For mitochondrial proteins, avoid freeze-thaw cycles that may degrade mitochondrial structures .

• Protein loading: Increase protein loading to 30-50 μg per lane for cell lysates and 50-75 μg for tissue samples when detecting endogenous ARMC10 .

• Antibody concentration adjustment: Increase primary antibody concentration to 1:500 ratio and extend incubation time to overnight at 4°C .

• Enhanced chemiluminescence: Use high-sensitivity ECL substrates for detection, as endogenous ARMC10 may be expressed at moderate levels in some cell types .

• Positive control inclusion: Include positive control samples (A431 cells, HEK-293 cells, or brain tissue lysates) where ARMC10 expression has been confirmed .

What strategies can resolve high background issues in ARMC10 immunostaining?

To minimize background in immunostaining:

• Blocking optimization: Extend blocking time to 1-2 hours using 5% BSA or 10% normal serum from the same species as the secondary antibody .

• Antibody dilution: Increase dilution of primary antibody to 1:200-1:500 for immunohistochemistry and 1:100-1:500 for immunofluorescence .

• Secondary antibody controls: Include controls with secondary antibody only to identify non-specific binding.

• Washing optimization: Perform more extensive washing steps (5-6 times for 5 minutes each) with 0.1% Tween-20 in PBS.

• Autofluorescence reduction: For tissues with high autofluorescence, pretreat sections with 0.1% Sudan Black B in 70% ethanol for 20 minutes before antibody incubation .