atp6v1c1b Antibody

What is ATP6V1C1 and what cellular functions does it perform?

ATP6V1C1 is a 42kDa protein that functions as an essential component of the vacuolar ATPase (V-ATPase) proton pump. It plays a crucial role in extracellular acidification, particularly at the osteoclast ruffled border where it contributes to bone resorption processes. Research has demonstrated that ATP6V1C1 is highly expressed in osteoclasts where it interacts with Atp6v0a3 (a3) and localizes primarily to the ruffled border in activated osteoclasts. Beyond its role in the proton pump assembly, ATP6V1C1 appears to regulate F-actin ring formation, which is essential for osteoclast activation and function . The protein consists of 382 amino acids with a calculated molecular weight of 44 kDa, though it is typically observed at 42-44 kDa in experimental contexts .

How does ATP6V1C1 expression differ across tissues and what implications does this have for antibody selection?

Expression analysis reveals that ATP6V1C1 is ubiquitously expressed across various tissues but with significant variation in expression levels. Microarray data indicates ATP6V1C1 is expressed approximately 139-fold higher than ATP6V1C2 in osteoclasts and 42-fold higher in osteoclasts compared to stromal cells . Semi-quantitative RT-PCR assays have detected ATP6V1C1 expression in brain, kidney, and testis, though at lower levels than in osteoclasts. In contrast, the isoforms ATP6V1C2a and ATP6V1C2b show tissue-specific expression patterns, with C2-a predominantly in lung and C2-b in kidney . When selecting antibodies for cross-reactivity, researchers should consider these tissue-specific expression patterns and choose antibodies validated for their specific experimental system to ensure accurate detection of the target protein.

What are the most reliable validation methods for ATP6V1C1 antibodies?

Validation of ATP6V1C1 antibodies should employ multiple complementary approaches. Western blotting serves as a primary validation method, with antibodies demonstrating specific detection of bands at the expected molecular weight (42-44 kDa). Knockdown or knockout models provide compelling validation - for example, siRNA experiments targeting ATP6V1C1 have shown effective depletion of protein expression with corresponding reduction in antibody signal . Positive sample validation is equally important, with antibody reactivity confirmed across multiple validated tissues and cell lines known to express ATP6V1C1, such as HeLa cells, NIH/3T3 cells, human brain tissue, or osteoclast cultures . Immunohistochemistry can provide additional validation by demonstrating appropriate subcellular localization, such as enrichment at the ruffled border in activated osteoclasts .

What are the optimal applications and dilutions for ATP6V1C1 antibodies?

ATP6V1C1 antibodies have been successfully employed in multiple experimental approaches with application-specific optimal dilutions. For Western blotting, dilutions ranging from 1:1000 to 1:4000 have yielded specific detection of ATP6V1C1 . Immunohistochemistry applications typically require higher antibody concentrations, with recommended dilutions between 1:20 and 1:200 . The exact dilution should be optimized for each experimental system and antibody lot. For certain applications like immunofluorescence or ELISA, researchers may need to conduct titration experiments to determine optimal conditions. The choice of application should align with research objectives - Western blotting for protein expression quantification, IHC for localization studies, and techniques like immunoprecipitation for protein interaction analyses.

What methodology should be employed for sample preparation to enhance ATP6V1C1 detection?

Sample preparation significantly impacts ATP6V1C1 detection quality. For Western blotting, efficient lysis buffers containing both ionic and non-ionic detergents facilitate extraction of membrane-associated proteins like ATP6V1C1. Including protease inhibitors prevents degradation during sample processing. For immunohistochemistry, different antigen retrieval methods may be necessary - reports indicate that TE buffer at pH 9.0 provides effective retrieval for ATP6V1C1 antibodies, though citrate buffer at pH 6.0 has been suggested as an alternative . When working with osteoclasts, timing is critical, as ATP6V1C1 expression increases significantly after RANKL induction, reaching peak levels around day 4-7 . For optimal results, researchers should harvest osteoclasts during this window when studying ATP6V1C1 expression or function.

How can researchers effectively use ATP6V1C1 antibodies in knockdown validation studies?

Knockdown validation using ATP6V1C1 antibodies requires careful experimental design. Lentiviral-mediated siRNA delivery has proven effective for ATP6V1C1 knockdown in primary osteoclast cultures. When designing such experiments, multiple siRNA sequences should be tested, as efficiency varies significantly - in published studies, some siRNAs achieved only 27-34% knockdown while others reached 98.8% depletion . Western blotting with ATP6V1C1 antibodies provides quantitative assessment of knockdown efficiency. Including appropriate controls is essential: non-targeting siRNA (e.g., against LacZ) and untreated controls establish baseline expression, while targeting related genes (such as Atp6v0a3) helps distinguish specific from non-specific effects . Functional assays following knockdown can reveal physiological implications, such as the observed impairment of acidification activity and bone resorption in ATP6V1C1-depleted osteoclasts .

How should researchers interpret variations in ATP6V1C1 band patterns in Western blots?

When interpreting Western blot results with ATP6V1C1 antibodies, researchers should anticipate a primary band at 42-44 kDa representing the full-length protein . Variations in band patterns may reflect biological phenomena rather than technical artifacts. Post-translational modifications such as phosphorylation can cause slight molecular weight shifts. The presence of multiple bands may indicate splice variants, protein degradation, or cross-reactivity with related proteins like ATP6V1C2 isoforms. To distinguish between these possibilities, researchers should include appropriate controls such as recombinant proteins and lysates from tissues with known expression profiles. Comparative analysis across multiple experimental conditions and antibodies targeting different epitopes can help validate band identity. Quantification should focus on bands of the expected molecular weight, with normalization to appropriate loading controls to account for sample variation.

What approaches can reveal ATP6V1C1's interactions with other V-ATPase components?

Investigating ATP6V1C1's interactions with other V-ATPase components requires a multifaceted approach. Co-immunoprecipitation using ATP6V1C1 antibodies can pull down interacting proteins, with reciprocal experiments using antibodies against suspected partners like Atp6v0a3 providing validation . Proximity ligation assays offer in situ visualization of protein interactions within intact cells. For spatial relationships, co-localization studies using immunofluorescence microscopy with ATP6V1C1 antibodies and markers for cellular structures provide valuable information - research has demonstrated co-localization of ATP6V1C1 with microtubules in the plasma membrane vicinity and with F-actin in the cytoplasm of mature osteoclasts . Functional interaction studies can employ knockout or knockdown approaches targeting individual components while monitoring effects on others. Biochemical assays measuring V-ATPase activity following manipulation of ATP6V1C1 levels provide functional confirmation of these interactions.

How can ATP6V1C1 antibodies be utilized to investigate its role in F-actin ring formation?

ATP6V1C1's role in F-actin ring formation represents a significant research area amenable to antibody-based investigations. Immunofluorescence co-staining with ATP6V1C1 antibodies and F-actin markers (such as phalloidin) can reveal spatial relationships between these components, with studies showing that ATP6V1C1 co-localizes with F-actin primarily at the cell periphery in mature osteoclasts . Live-cell imaging using fluorescently tagged ATP6V1C1 antibody fragments might enable temporal tracking of its association with F-actin during osteoclast activation. Comparative analyses between ATP6V1C1-depleted and control osteoclasts demonstrate that ATP6V1C1 knockdown severely impairs F-actin ring formation, unlike knockdown of other V-ATPase components such as a3 . This suggests a unique role for ATP6V1C1 in cytoskeletal organization beyond its function in the proton pump. Researchers can further dissect this relationship through domain mapping experiments using truncated ATP6V1C1 constructs and corresponding antibodies to identify regions critical for F-actin interaction.

What are common troubleshooting strategies when ATP6V1C1 antibodies yield inconsistent results?

When encountering inconsistent results with ATP6V1C1 antibodies, researchers should systematically evaluate multiple parameters. For Western blotting applications, sample quality significantly impacts results - degraded samples may show multiple bands or weak signals. Optimization of protein extraction using buffers containing appropriate detergents and protease inhibitors can improve detection. For immunohistochemistry, antigen retrieval methods critically affect epitope accessibility - if standard citrate buffer (pH 6.0) yields poor results, alternative methods like TE buffer at pH 9.0 should be tested . Antibody concentration requires careful titration, as both insufficient antibody (weak signal) and excess antibody (high background) compromise results. Storage conditions affect antibody performance; ATP6V1C1 antibodies are typically stable for one year at -20°C, though aliquoting may be necessary for smaller quantities to prevent freeze-thaw cycles . Cross-reactivity with related proteins may occur, particularly between ATP6V1C1 and ATP6V1C2 isoforms, necessitating validation in systems with known expression profiles.

How can researchers quantitatively assess ATP6V1C1 expression across different experimental conditions?

Quantitative assessment of ATP6V1C1 expression requires rigorous methodological approaches. Western blotting with ATP6V1C1 antibodies followed by densitometric analysis enables relative quantification when normalized to appropriate loading controls such as housekeeping proteins. For more precise quantification, researchers should employ standard curves using recombinant ATP6V1C1 protein. RT-qPCR complements protein-level analyses by measuring ATP6V1C1 mRNA expression, though post-transcriptional regulation may cause discrepancies between mRNA and protein levels. Time-course experiments are particularly informative - studies have shown ATP6V1C1 protein levels in osteoclasts increase through day 4 of RANKL/M-CSF treatment and remain elevated through day 7 . This temporal profile should guide experimental design when comparing conditions. For cellular heterogeneity, flow cytometry with ATP6V1C1 antibodies can quantify expression at the single-cell level, while immunohistochemistry with digital image analysis provides spatial information within tissue contexts.

What novel research applications are emerging for ATP6V1C1 antibodies beyond traditional methods?

Emerging applications for ATP6V1C1 antibodies extend beyond conventional techniques. Proximity-dependent biotinylation (BioID) or APEX2 approaches using ATP6V1C1 fusion proteins followed by antibody-based detection enable systematic mapping of the ATP6V1C1 interactome in living cells. Super-resolution microscopy with ATP6V1C1 antibodies provides nanoscale localization details, potentially revealing previously undetected structural arrangements within the V-ATPase complex. Antibody-based proteomics techniques like reverse phase protein arrays (RPPA) allow high-throughput screening of ATP6V1C1 expression across numerous samples. For therapeutic applications, ATP6V1C1 antibodies conjugated to nanoparticles or drug-delivery systems could target cells with aberrant ATP6V1C1 expression. In disease contexts, particularly bone disorders involving osteoclast dysfunction, ATP6V1C1 antibodies may serve as diagnostic tools in immunohistochemical panels. As gene therapy approaches advance, antibodies will play crucial roles in validating the expression and function of modified ATP6V1C1 proteins in preclinical models.

What methodological approaches best reveal ATP6V1C1's role in cellular acidification and bone resorption?

Investigating ATP6V1C1's role in cellular acidification and bone resorption requires complementary methodological approaches. Functional acidification assays using pH-sensitive dyes in conjunction with ATP6V1C1 knockdown or overexpression provide direct evidence of its contribution to proton transport. Bone resorption can be quantitatively assessed using dentine or synthetic substrate assays, measuring resorption pit formation in control versus ATP6V1C1-depleted osteoclasts . Immunofluorescence with ATP6V1C1 antibodies confirms its localization to the ruffled border in activated osteoclasts, the site of active proton secretion during bone resorption . For in vivo relevance, histomorphometric analysis of bone parameters in animal models with manipulated ATP6V1C1 expression reveals physiological significance. Combining these approaches with molecular interventions targeting specific ATP6V1C1 domains can map functional regions responsible for acidification versus cytoskeletal organization. Time-lapse microscopy with simultaneous pH measurement and ATP6V1C1 visualization could further elucidate the temporal relationship between ATP6V1C1 localization and acidification events.

What technological advancements might enhance the utility of ATP6V1C1 antibodies in future research?

Future technological advancements will expand ATP6V1C1 antibody applications in several directions. Development of conformation-specific antibodies could distinguish between ATP6V1C1's assembly states within the V-ATPase complex, providing insights into its activation mechanisms. Site-specific phospho-antibodies targeting regulatory residues would enable monitoring of ATP6V1C1's post-translational modifications in response to cellular signaling. Advances in cryo-electron microscopy will benefit from highly specific ATP6V1C1 antibodies to stabilize complexes for structural determination at atomic resolution. For clinical applications, development of humanized ATP6V1C1 antibodies could enable therapeutic targeting in diseases involving aberrant osteoclast activity. Integration of machine learning algorithms with digital pathology using ATP6V1C1 antibodies might improve diagnostic precision in bone disorders. Finally, emerging spatial transcriptomics and proteomics technologies combining RNA sequencing with antibody-based protein detection will provide unprecedented insights into ATP6V1C1's expression patterns and functional relationships at the single-cell level within complex tissues.