ASIC5 Antibody

What is ASIC5 and why is it significant in research?

ASIC5 (Acid-Sensing Ion Channel 5) is a cation channel that exhibits selectivity for sodium and is inhibited by amiloride. It gives rise to very low constitutive currents in the absence of activation and functions as a multi-pass membrane protein. The significance of ASIC5 in research stems from its distinct tissue expression pattern, being primarily detected in the small intestine (duodenum and jejunum) with very low levels in testis and rectum . Additionally, mouse studies have revealed that ASIC5 is restrictively expressed in interneurons in the granular layer of the vestibulocerebellum, particularly in lobules X, IXb, and IXc, suggesting specific neurological functions . Understanding ASIC5 may provide insights into intestinal ion transport mechanisms and certain neurological processes, making it an important target for researchers investigating gastrointestinal physiology and cerebellar function.

Which animal models are suitable for ASIC5 antibody validation?

For ASIC5 antibody validation, mouse and rat models represent the most well-established and suitable experimental systems. Commercial antibodies are specifically reactive to mouse and rat ASIC5 proteins, as indicated in product specifications . The Asic5 reporter mouse model (Asic5 tm2a(KOMP)Wtsi) has been particularly valuable for studying ASIC5 expression patterns. This model uses a gene trapping cassette with β-galactosidase activity to report ASIC5 expression, allowing visualization of ASIC5-expressing cells through β-galactosidase staining . When validating ASIC5 antibodies, researchers should incorporate appropriate positive controls (tissues known to express ASIC5, such as cerebellum and small intestine) and negative controls (tissues with minimal expression or tissues from ASIC5 knockout animals). Additionally, validation should include multiple techniques (e.g., Western blot, immunohistochemistry) to confirm antibody specificity across different experimental conditions.

What are the recommended applications for ASIC5 antibodies?

ASIC5 antibodies are versatile research tools with several validated applications for investigating this ion channel. The primary recommended applications include Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), and Immunohistochemistry (IHC) . For Western blot applications, optimal dilution ranges typically fall between 1:500-1:2000, though researchers should determine the optimal concentration for their specific experimental conditions . For ELISA applications, a starting concentration of 1 μg/mL is recommended, with optimization based on specific assay requirements. For immunohistochemistry, a dilution range of 1:20-1:200 is suggested . These applications allow researchers to detect and quantify ASIC5 protein levels in various tissues, assess expression patterns in histological samples, and investigate protein-protein interactions. Researchers should always validate antibody performance in their specific experimental systems, as performance can vary across different tissue preparations and experimental conditions.

How should ASIC5 antibodies be stored to maintain optimal activity?

For optimal maintenance of ASIC5 antibody activity, proper storage is essential. ASIC5 antibodies are typically formulated in PBS with 0.02% Sodium Azide and 50% Glycerol at pH 7.3 . The recommended storage temperature is -20°C, where antibodies can be maintained for up to one year from the date of receipt . It is crucial to avoid repeated freeze-thaw cycles, as these can lead to denaturation and degradation of antibody proteins, compromising specificity and sensitivity. For working solutions, aliquoting the stock antibody into smaller volumes before freezing is advisable to minimize freeze-thaw cycles. When handling antibodies, researchers should use clean, nuclease-free tubes and pipette tips to prevent contamination. For short-term storage during experiments, antibodies can be kept at 4°C for several days, but should be returned to -20°C for long-term storage. Always check the manufacturer's specific recommendations, as formulations may vary slightly between suppliers.

How can I optimize ASIC5 antibody conditions for detecting low expression levels in neural tissues?

Optimizing ASIC5 antibody protocols for detecting low expression levels in neural tissues requires a systematic approach to amplify signal while maintaining specificity. First, consider implementing a signal amplification method such as tyramide signal amplification (TSA) or avidin-biotin complex (ABC) enhancement, which can increase sensitivity by 10-100 fold. Antigen retrieval optimization is also critical; for ASIC5 in neural tissues, heat-induced epitope retrieval using citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0) should be empirically tested to determine which best exposes the epitope of interest . Increase the primary ASIC5 antibody incubation time to 48-72 hours at 4°C with gentle agitation to improve penetration into neural tissues. Additionally, the use of background-reducing agents such as 1-5% BSA, normal serum matching the secondary antibody host, or commercial background reducers can significantly improve signal-to-noise ratio. Reference the restrictive expression pattern of ASIC5 in the vestibulocerebellum, particularly in interneurons of lobules X, IXb, and IXc, to properly target your analysis . Finally, implement a tiered dilution series (e.g., 1:20, 1:50, 1:100, 1:200) with extended development times to empirically determine the optimal working concentration for your specific tissue samples.

What are the key considerations when comparing data from different ASIC5 antibodies?

When comparing data generated using different ASIC5 antibodies, researchers must account for several critical variables to ensure valid interpretations. First, epitope differences must be considered; antibodies targeting different regions of ASIC5 (such as the immunogen region 140-330 versus other domains) may yield different staining patterns or intensities due to epitope accessibility, post-translational modifications, or protein interactions . Host species differences can introduce variability; rabbit polyclonal antibodies may have different background staining patterns compared to antibodies raised in other species . Clonality is another important factor; polyclonal antibodies recognize multiple epitopes and may provide stronger signals but with potentially higher background compared to monoclonal antibodies. Validation methodology should be scrutinized; antibodies validated using different techniques (WB vs. IHC) may perform differently in your specific application . For quantitative comparisons, establish normalization methods using housekeeping proteins and include positive controls (tissues with known ASIC5 expression like small intestine or vestibulocerebellum) and negative controls (tissues with minimal expression or ASIC5 knockout samples) across all experiments. Finally, implement side-by-side testing of antibodies under identical conditions on the same tissue samples to directly assess performance differences and document these comparative analyses in your methods section to ensure reproducibility.

How can I differentiate between ASIC5 and other ASIC family members in experimental settings?

Differentiating between ASIC5 and other ASIC family members in experimental settings requires a multi-faceted approach to ensure specificity. First, select antibodies raised against unique regions of ASIC5 that have minimal sequence homology with other ASIC family members. The immunogen region 140-330 of human ASIC5 (NP_059115.1) has been used successfully for antibody generation . Implement rigorous antibody validation using tissues from knockout models; the Asic5 tm2a(KOMP)Wtsi mouse model provides an excellent negative control for validating ASIC5-specific antibodies . Conduct pre-absorption controls by incubating the antibody with excess purified ASIC5 peptide before immunostaining to confirm specificity. For more definitive characterization, perform side-by-side analysis using multiple antibodies targeting different epitopes of ASIC5. At the mRNA level, design primers for RT-PCR or in situ hybridization that target unique regions of ASIC5 transcripts not shared with other family members. Leverage the distinct tissue expression pattern of ASIC5 (predominantly in small intestine, duodenum, and jejunum, with restricted expression in cerebellar interneurons) as a reference point for comparison with other ASIC family members . Finally, functional differentiation can be achieved using patch-clamp electrophysiology with amiloride as an inhibitor, as ASIC5 shows a characteristic response to this compound that may differ from other family members.

What approaches can resolve contradictory findings in ASIC5 expression patterns across studies?

Resolving contradictory findings in ASIC5 expression patterns requires systematic investigation of methodological differences and biological variables. First, perform a comprehensive meta-analysis of published studies, categorizing them by detection method (antibody-based versus reporter gene approaches), species differences, and specific tissues examined. The restrictive expression of ASIC5 in interneurons of the vestibulocerebellum in mouse models suggests highly specific localization that might be missed in broader surveys . Implement parallel methodologies on identical samples; for example, compare immunohistochemistry with in situ hybridization and RT-PCR on the same tissue specimens to cross-validate findings. Consider developmental timing as a source of variability, as expression patterns may change during different developmental stages. Replicate contradictory findings using identical protocols but in independent laboratories to determine whether results are reproducible. Investigate strain-specific differences in animal models, as genetic background can significantly influence gene expression patterns. Employ single-cell RNA sequencing to resolve cell type-specific expression that might be diluted in whole-tissue analyses. For antibody-based studies specifically, validate antibody specificity using knockout tissues and pre-absorption controls to eliminate false positives. Finally, explicitly test hypotheses about the source of contradictions; for example, if two studies show different expression in cerebellum, design experiments that specifically address variables like fixation methods, detection sensitivity, or region-specific sampling that might explain the discrepancies.

What is the optimal sample preparation protocol for ASIC5 immunohistochemistry in brain tissue?

The optimal sample preparation protocol for ASIC5 immunohistochemistry in brain tissue requires careful attention to fixation, antigen retrieval, and blocking procedures. For fixation, perfuse the animal with ice-cold PBS followed by 4% paraformaldehyde in PBS (pH 7.4). Post-fix the extracted brain for 24-48 hours at 4°C, then transfer to 30% sucrose in PBS for cryoprotection until the tissue sinks. For paraffin embedding, dehydrate in graded ethanol series, clear in xylene, and embed in paraffin. For frozen sections, embed in OCT compound and freeze at -80°C. Cut sections at 5-10 μm thickness for optimal ASIC5 detection, particularly when targeting the vestibulocerebellum where ASIC5 expression is concentrated in interneurons of lobules X, IXb, and IXc . For antigen retrieval, heat-mediated retrieval in citrate buffer (pH 6.0) for 20 minutes at 95-100°C yields optimal results for ASIC5 epitope exposure. Block sections with 5% normal serum (matching the host species of the secondary antibody) plus 1% BSA and 0.3% Triton X-100 in TBS for 1-2 hours at room temperature. Apply primary ASIC5 antibody at dilutions between 1:20-1:200 , incubating overnight at 4°C in a humidified chamber. For detection, use biotinylated secondary antibody followed by avidin-biotin complex and DAB visualization, or fluorophore-conjugated secondary antibodies for immunofluorescence. Include positive controls (cerebellum sections from wild-type animals) and negative controls (cerebellum sections from Asic5 tm2a(KOMP)Wtsi knockout mice or primary antibody omission) to validate staining specificity .

What are the recommended Western blot protocols for detecting ASIC5 in different tissue samples?

The recommended Western blot protocol for detecting ASIC5 across different tissue samples requires optimization for this specific ion channel. Begin with sample preparation by homogenizing tissue in RIPA buffer supplemented with protease inhibitors, phosphatase inhibitors, and 1mM EDTA. For brain tissue, particularly cerebellum where ASIC5 shows restrictive expression in interneurons of the vestibulocerebellum , use a more gentle lysis buffer (25mM Tris-HCl pH 7.4, 150mM NaCl, 1mM EDTA, 1% NP-40, 5% glycerol) to preserve membrane protein integrity. For intestinal samples (duodenum, jejunum) where ASIC5 is more abundantly expressed , include additional mechanical disruption steps to overcome the tough tissue matrix. Separate proteins on 10-12% SDS-PAGE gels, loading 30-50μg of total protein per lane. Transfer to PVDF membranes (preferred over nitrocellulose for membrane proteins) at 25V overnight at 4°C for optimal transfer of membrane-bound proteins like ASIC5. Block with 5% non-fat dry milk in TBST for 1 hour at room temperature. Incubate with primary ASIC5 antibody at dilutions of 1:500-1:2000 in blocking buffer overnight at 4°C with gentle agitation. After washing, incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature. Develop using enhanced chemiluminescence with extended exposure times (up to 10 minutes) to detect potentially low signals. Include positive controls (small intestine lysate) and negative controls (tissue from ASIC5 knockout animals) to validate antibody specificity. The expected molecular weight for human ASIC5 is approximately 50-55 kDa, though this may vary slightly between species.

How should co-localization studies with ASIC5 and neuronal markers be designed?

Co-localization studies with ASIC5 and neuronal markers require careful experimental design to yield accurate and interpretable results. Begin by selecting appropriate neuronal markers based on the brain region and cell type of interest; for studies in the vestibulocerebellum where ASIC5 expression is restrictively localized in interneurons of lobules X, IXb, and IXc , consider markers such as calbindin (for Purkinje cells), parvalbumin (for basket and stellate cells), or NeuN (for most neurons). Prepare tissue sections using perfusion-fixation with 4% paraformaldehyde and cut at 20-30μm thickness to allow adequate penetration of antibodies while maintaining cellular morphology. Implement sequential immunostaining protocols when antibodies are raised in the same host species; complete one staining cycle, fix with 4% paraformaldehyde for 10 minutes, then proceed with the second immunostaining sequence. For simultaneous detection, select ASIC5 antibodies and neuronal marker antibodies raised in different host species to avoid cross-reactivity. Use different fluorophores with minimal spectral overlap (e.g., Alexa 488 and Alexa 594) for clear distinction between signals. Include appropriate controls: single-labeled samples to assess bleed-through, secondary-only controls to assess non-specific binding, and absorption controls for both ASIC5 and neuronal marker antibodies. Acquire images using confocal microscopy with sequential scanning to prevent cross-channel contamination, and maintain consistent acquisition parameters across all samples. For quantitative co-localization analysis, use established metrics such as Pearson's correlation coefficient, Manders' overlap coefficient, or intensity correlation analysis, and report these values alongside representative images.

What are the critical controls needed for validating ASIC5 antibody specificity?

Validating ASIC5 antibody specificity requires a comprehensive set of controls to ensure reliable experimental outcomes. First, genetic controls are essential: tissues from Asic5 tm2a(KOMP)Wtsi knockout mice provide the gold standard negative control, while tissues with confirmed ASIC5 expression (vestibulocerebellum, small intestine) serve as positive controls . Peptide competition/pre-absorption controls should be performed by pre-incubating the ASIC5 antibody with excess immunizing peptide (the 140-330 amino acid region of human ASIC5) before application to samples; specific staining should be eliminated or significantly reduced. Cross-reactivity assessment is critical; test the antibody on tissues known to express other ASIC family members but not ASIC5 to confirm absence of cross-reactivity. Implement multiple detection methods validation by comparing results from different techniques (Western blot, IHC, ICC) using the same antibody to confirm consistent detection patterns. For antibody dilution gradients, perform a systematic dilution series (e.g., 1:20, 1:50, 1:100, 1:200 for IHC; 1:500, 1:1000, 1:2000 for WB) to determine the optimal signal-to-noise ratio. Secondary antibody-only controls (omitting primary ASIC5 antibody) are essential to identify non-specific binding of the secondary antibody. Host tissue matrix controls involve applying the antibody to tissues from the same host species as the antibody was raised in (typically rabbit for ASIC5 antibodies) to identify potential tissue-specific background. Batch validation should be performed by testing each new lot of antibody alongside a previously validated lot to ensure consistent performance over time.

How might ASIC5 antibodies contribute to understanding its role in neurological disorders?

ASIC5 antibodies may significantly advance our understanding of neurological disorders through targeted investigation of this channel's function in specific brain regions. Given the restrictive expression of ASIC5 in interneurons of the vestibulocerebellum (lobules X, IXb, and IXc) , researchers should prioritize investigating disorders affecting cerebellar function and motor coordination. ASIC5 antibodies could enable comparative immunohistochemical studies between healthy controls and patients with cerebellar ataxias, vestibular disorders, or other conditions affecting the vestibulocerebellum. These antibodies would facilitate detailed mapping of expression changes in disease states, potentially identifying ASIC5 dysregulation as a contributing factor to pathogenesis. Researchers could employ ASIC5 antibodies in co-immunoprecipitation experiments to identify novel protein-protein interactions that might be altered in neurological conditions, revealing previously unknown signaling pathways. For clinical applications, developing phospho-specific ASIC5 antibodies could help determine if post-translational modifications of this channel correlate with disease progression or severity. In animal models of cerebellar dysfunction, ASIC5 antibodies would enable precise tracking of expression changes throughout disease progression and in response to therapeutic interventions. Furthermore, ASIC5 antibodies with neutralizing capacity could serve as experimental tools to temporarily block channel function in specific brain regions, helping to elucidate its physiological role in neural circuits and potentially identifying new therapeutic targets for neurological disorders affecting cerebellar function.

What emerging techniques could enhance ASIC5 antibody-based research?

Emerging techniques offer significant potential to enhance ASIC5 antibody-based research, providing deeper insights into this ion channel's function and distribution. Proximity ligation assay (PLA) could revolutionize the study of ASIC5 protein interactions by enabling visualization of proteins that are within 40nm of each other, potentially revealing previously unknown binding partners and complexes in native tissue contexts. Expansion microscopy, which physically expands biological specimens while maintaining relative spatial relationships, would allow super-resolution imaging of ASIC5 distribution within the restrictively expressed regions of the vestibulocerebellum without requiring specialized microscopy equipment. CRISPR-epitope tagging could enable endogenous tagging of ASIC5 with small epitopes for antibody detection, avoiding potential artifacts associated with overexpression systems. Tissue clearing techniques (CLARITY, iDISCO, CUBIC) combined with light-sheet microscopy would permit whole-brain 3D mapping of ASIC5 expression at cellular resolution, providing comprehensive spatial context currently missing from conventional section-based approaches. Mass cytometry with metal-conjugated antibodies could enable simultaneous detection of ASIC5 alongside dozens of other markers in single cells, facilitating deep phenotyping of ASIC5-expressing neurons. For functional studies, optogenetic actuators could be expressed under the control of ASIC5 promoters (leveraging information from the Asic5 tm2a(KOMP)Wtsi mouse model) to specifically manipulate ASIC5-expressing neurons while simultaneously monitoring their activity with antibody-based detection of activity-dependent markers. Single-cell proteomics approaches could quantify ASIC5 expression levels in individual neurons, potentially revealing heterogeneity within seemingly uniform populations of vestibulocerebellar interneurons.

What strategies could improve next-generation ASIC5 antibody development?

Improving next-generation ASIC5 antibody development requires innovative strategies that enhance specificity, sensitivity, and versatility. Implementing epitope mapping through systematic analysis of the ASIC5 protein sequence would identify highly specific regions with minimal homology to other ASIC family members, particularly focusing beyond the currently used 140-330 amino acid region . Developing conformation-specific antibodies that recognize ASIC5 in its native, membrane-embedded form would significantly advance functional studies of this ion channel in its physiological context. Employing phage display technology with synthetic antibody libraries would generate highly specific monoclonal antibodies with potentially superior performance compared to traditional polyclonal approaches. Creating phospho-specific antibodies against predicted phosphorylation sites would enable studies of ASIC5 regulation through post-translational modifications. Developing nanobodies (single-domain antibodies) against ASIC5 would provide smaller probes with enhanced tissue penetration for imaging applications and potential intracellular expression as intrabodies for live-cell studies. Engineering bispecific antibodies that simultaneously target ASIC5 and a cell-type-specific marker would facilitate selective visualization of the channel in specific neuronal populations, particularly valuable for studying the restricted expression in vestibulocerebellar interneurons . Utilizing glycosylation-specific antibodies would enable detection of differentially glycosylated forms of ASIC5, potentially revealing functional subpopulations. Finally, implementing recombinant antibody production with site-specific conjugation would ensure batch-to-batch consistency and enable precise control over antibody modifications for specialized applications such as super-resolution microscopy.

What are the most significant recent advances in ASIC5 antibody research?

The most significant recent advances in ASIC5 antibody research have transformed our understanding of this unique ion channel's expression patterns and potential functions. The development of the Asic5 tm2a(KOMP)Wtsi reporter mouse model represents a breakthrough that has enabled precise cellular mapping of ASIC5 expression, revealing its highly restrictive localization in interneurons within the granular layer of the vestibulocerebellum, particularly in lobules X, IXb, and IXc . This model has provided critical negative control tissues for antibody validation while simultaneously offering insights into ASIC5's potential neurological functions. The generation of highly specific polyclonal antibodies against defined epitope regions (such as amino acids 140-330 of human ASIC5) has enhanced detection specificity across multiple experimental applications including Western blot, ELISA, and immunohistochemistry . These improved reagents have facilitated more reliable protein detection in tissues with low expression levels, such as testis and rectum. Methodological refinements in tissue preparation and antigen retrieval techniques have overcome previous challenges in detecting this membrane-bound ion channel, particularly in neural tissues where conventional fixation methods often masked epitope accessibility. The characterization of ASIC5's tissue distribution pattern, prominently in small intestine, duodenum, and jejunum , has directed research focus toward its potential roles in gastrointestinal physiology alongside its neurological functions. Together, these advances have established a solid foundation for future investigations into ASIC5's physiological roles and potential involvement in pathological conditions affecting the vestibulocerebellum and gastrointestinal system.