C10orf71 Antibody

What is C10orf71 and why is it important in cardiovascular research?

C10orf71 (Chromosome 10 Open Reading Frame 71) is a protein that has recently been identified as a causal gene for dilated cardiomyopathy. It is an intrinsically disordered protein specifically expressed in cardiomyocytes that appears to play a critical role in cardiac function. Structural analysis shows that C10orf71 has almost no regular secondary structure and exists in a disordered state throughout its entire length. The protein exhibits high hydrophilicity and is classified as unstable with an instability index of 65.67. C10orf71's expression increases significantly during heart development, similar to established cardiac markers like TNNT2, suggesting its involvement in the development and maturation of cardiac muscle. Recent research has demonstrated that loss-of-function variants in this gene are associated with dilated cardiomyopathy, making it an important target for cardiovascular research .

Where is C10orf71 protein expressed in human tissues?

C10orf71 shows highly specific expression patterns that are crucial for researchers to understand when designing experiments. It is highly and specifically expressed in human heart and skeletal muscle tissues. Single nucleus RNA sequencing data of human hearts demonstrates that C10orf71 is specifically expressed in cardiomyocytes, even more specifically than well-established cardiomyocyte markers such as TNNT2 and MYH7. Further analysis using published single-cell sequencing data reveals that C10orf71 is predominantly expressed in ventricular cardiomyocytes. Immunofluorescence studies have shown that C10orf71 colocalizes with markers of the Z disc (Actn2) and myofibers (Actc1) in mouse cardiomyocytes, a finding that has been validated in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) .

What are the primary applications for C10orf71 antibodies in research?

Currently available C10orf71 antibodies are primarily utilized for Western Blotting (WB) and ELISA applications. These antibodies allow researchers to detect C10orf71 protein expression in tissue samples and cell lysates, providing crucial data for studies investigating cardiac development, cardiomyopathies, and related cardiac pathologies. Given the specific expression pattern of C10orf71 in cardiomyocytes, these antibodies serve as valuable tools for studying cardiac-specific protein expression and localization. The antibodies can help validate genetic findings by confirming protein level changes that correspond to genetic variants, particularly in studies of dilated cardiomyopathy where C10orf71 has been implicated as a causal gene .

What types of C10orf71 antibodies are currently available for research?

Currently available C10orf71 antibodies include polyclonal rabbit antibodies that target specific amino acid regions of the protein. For example, one commercially available antibody (ABIN5025527) targets amino acids 609-658 of C10orf71 and is supplied as an unconjugated antibody that has been antigen affinity purified. These antibodies are typically provided in liquid format with buffers containing BSA and are specifically designed for research applications. They are validated for human reactivity and applicable in techniques such as Western blotting and ELISA. It's important to note that these antibodies are typically restricted for research use only, meaning they are not validated for diagnostic or therapeutic applications .

How can C10orf71 antibodies be used to investigate its role in dilated cardiomyopathy?

C10orf71 antibodies can be instrumental in investigating the protein's role in dilated cardiomyopathy through multiple approaches. Researchers can use these antibodies to compare C10orf71 protein expression and localization in tissue samples from healthy hearts versus DCM patients, particularly those with frameshift or loss-of-function variants in the C10orf71 gene. Immunofluorescence staining of cardiac tissue sections can reveal changes in C10orf71 distribution patterns, while co-immunoprecipitation experiments can identify protein interaction partners that might be disrupted in disease states. Additionally, Western blotting analysis can quantify changes in C10orf71 protein levels in patient samples or experimental models. These antibodies can also be used to validate CRISPR/Cas9 knockout models, as demonstrated in recent research where C10orf71-KO mice exhibited abnormal heart morphogenesis during embryonic development and cardiac dysfunction as adults. By employing C10orf71 antibodies in these contexts, researchers can gain insights into how loss of this protein leads to contractile dysfunction and ultimately dilated cardiomyopathy .

What are the optimal conditions for detecting C10orf71 in cardiomyocyte samples?

For optimal detection of C10orf71 in cardiomyocyte samples, researchers should consider several technical factors based on the protein's unique characteristics and expression patterns. Since C10orf71 is an intrinsically disordered protein with high hydrophilicity and instability (index of 65.67), sample preparation must be carefully controlled to prevent protein degradation. Fresh tissue samples or cells should be processed rapidly and maintained at cold temperatures throughout lysate preparation. For Western blotting, reducing conditions are typically recommended, with separation on 8-10% SDS-PAGE gels to accommodate the protein's size. When performing immunofluorescence, co-staining with established cardiomyocyte markers such as Actn2 (Z-disc) or Actc1 (myofibers) can help confirm specific detection, as C10orf71 has been shown to colocalize with these structures. The working dilution should be empirically determined for each experimental setup, but manufacturers typically recommend starting with standard dilutions for Western blotting and adjusting based on signal intensity. For immunoprecipitation studies, native conditions may need to be optimized to preserve protein-protein interactions involving this disordered protein .

How can researchers validate C10orf71 antibody specificity in experimental models?

Validating C10orf71 antibody specificity is critical for ensuring reliable experimental results, especially given the recent characterization of this protein. Several complementary approaches are recommended for thorough validation. First, researchers should perform Western blotting with samples from tissues known to express C10orf71 (heart and skeletal muscle) alongside negative control tissues where the protein is not expressed. A specific antibody will show bands of the expected molecular weight only in positive samples. Second, antibody specificity can be confirmed using genetic models such as CRISPR/Cas9-generated C10orf71 knockout mice or cells, where the antibody signal should be absent or significantly reduced. This approach was successfully used in recent research to confirm knockout efficiency in C10orf71-KO mice. Third, for polyclonal antibodies, pre-absorption controls using the immunizing peptide can verify specific binding. Finally, orthogonal methods such as RNA interference or overexpression of tagged C10orf71 constructs can provide additional confirmation of antibody specificity. For advanced validation, mass spectrometry analysis of immunoprecipitated proteins can identify whether the antibody is capturing the intended target without significant cross-reactivity .

What methodological approaches can be used to study C10orf71 protein interactions?

To study C10orf71 protein interactions, researchers can employ several methodological approaches tailored to this intrinsically disordered protein. Co-immunoprecipitation (Co-IP) using C10orf71 antibodies represents a primary approach, allowing for the pull-down of protein complexes that include C10orf71 and its binding partners. Since C10orf71 colocalizes with Z-disc and myofiber markers in cardiomyocytes, targeted Co-IP experiments with known sarcomeric proteins might reveal functional interactions. Proximity ligation assays (PLA) offer another approach to visualize protein-protein interactions in situ, providing spatial information about where interactions occur within cardiomyocytes. For more comprehensive interaction mapping, BioID or APEX2 proximity labeling can be employed by fusing C10orf71 to a biotin ligase, enabling identification of the complete protein interaction network. Given C10orf71's role in contractile function and its effects on cardiac gene expression and splicing when knocked out, particular attention should be paid to interactions with contractile apparatus components and RNA processing machinery. Recent research has demonstrated that C10orf71-null cardiomyocytes exhibit impaired contractile function without affecting sarcomere structure, suggesting that C10orf71 might interact with proteins involved in excitation-contraction coupling rather than structural components .

What are the best practices for Western blotting with C10orf71 antibodies?

For optimal Western blotting results with C10orf71 antibodies, researchers should implement several specific practices tailored to this protein's characteristics. Sample preparation should include protease inhibitors to prevent degradation of this unstable protein (instability index of 65.67). For gel electrophoresis, 8-10% polyacrylamide gels are recommended to properly resolve C10orf71. During transfer, a PVDF membrane may provide better protein retention than nitrocellulose. For blocking, 5% non-fat dry milk in TBST is generally effective, though BSA-based blocking solutions might reduce background for certain antibody lots. When using the commercially available polyclonal antibody (e.g., ABIN5025527), start with the manufacturer's recommended dilution and optimize empirically for your specific sample type. Include positive controls (heart or skeletal muscle lysates) and negative controls (tissues not expressing C10orf71) in each experiment. For detecting truncated C10orf71 variants, as might be present in dilated cardiomyopathy patients with frameshift mutations, researchers should consider the epitope location of the antibody (e.g., amino acids 609-658) and whether this region would be present in the truncated protein. Finally, longer exposure times may be necessary when detecting endogenous C10orf71 in complex samples due to its relatively low abundance compared to major structural proteins in cardiomyocytes .

How should immunofluorescence protocols be optimized for C10orf71 detection?

Optimizing immunofluorescence protocols for C10orf71 detection requires careful attention to fixation, permeabilization, and antibody incubation conditions. For cardiac tissue sections or cultured cardiomyocytes, 4% paraformaldehyde fixation for 10-15 minutes at room temperature preserves the native localization of C10orf71 while maintaining tissue morphology. Permeabilization with 0.2% Triton X-100 for 10 minutes allows antibody access to intracellular targets without disrupting the protein's association with sarcomeric structures. Since C10orf71 colocalizes with Z-disc (Actn2) and myofiber (Actc1) markers, co-staining with these established markers provides internal validation of staining patterns. Use longer primary antibody incubation times (overnight at 4°C) with gentle agitation to enhance antibody penetration. For polyclonal C10orf71 antibodies, a 1:100 to 1:200 dilution range typically provides good signal-to-noise ratio, though this should be empirically determined. Include a nuclear counterstain like DAPI to facilitate cell identification. When imaging, confocal microscopy with z-stack acquisition is recommended to accurately assess the colocalization of C10orf71 with sarcomeric structures. For quantitative analyses, consistent exposure settings across all experimental conditions are essential, and image analysis should include sarcomere length measurements to account for contraction state variations .

What controls should be included when using C10orf71 antibodies in research?

When using C10orf71 antibodies in research, a comprehensive set of controls should be included to ensure experimental validity and interpretability. First, include positive tissue controls known to express C10orf71 (heart and skeletal muscle) and negative tissue controls (tissues without C10orf71 expression) to confirm antibody specificity. For genetic studies involving C10orf71 variants, wild-type samples should be processed alongside mutant samples under identical conditions. When feasible, include knockout or knockdown controls where C10orf71 has been genetically depleted, as was done in studies using C10orf71-KO mice. For immunofluorescence experiments, include a secondary antibody-only control to assess non-specific binding, and an isotype control antibody to evaluate background staining. In co-localization studies, single-color controls are essential for setting up proper imaging parameters and addressing potential spectral overlap. For experiments investigating truncated C10orf71 variants resulting from frameshift mutations, controls expressing known truncated forms can help validate antibody detection capabilities. Finally, when investigating disease models, age-matched and sex-matched controls should be used to account for potential confounding factors in C10orf71 expression or localization .

How can researchers quantify C10orf71 levels in cardiac samples?

Researchers can employ multiple complementary methods to quantify C10orf71 levels in cardiac samples with high precision and reliability. For protein-level quantification, quantitative Western blotting represents a primary approach, using chemiluminescent or fluorescent detection systems with appropriate standard curves. Densitometric analysis should be normalized to both total protein loading (via stain-free gels or housekeeping proteins stable in cardiac contexts) and to cardiomyocyte-specific markers to account for variations in cardiomyocyte content across samples. Enzyme-linked immunosorbent assays (ELISA) using available C10orf71 antibodies provide another quantitative method for protein measurement in tissue lysates. For spatial quantification, immunofluorescence combined with digital image analysis allows measurement of C10orf71 signal intensity and distribution patterns within cardiomyocytes, which can be particularly valuable when assessing diseased tissues. At the transcript level, quantitative PCR or RNA sequencing provides insight into C10orf71 expression, though transcript levels may not perfectly correlate with protein abundance, especially for genes producing intrinsically disordered proteins like C10orf71. When analyzing patient samples or disease models with frameshift mutations, researchers should consider using multiple antibodies targeting different epitopes to ensure detection of potential truncated products, as demonstrated in studies where frameshift variant mRNA levels were significantly decreased compared to wild-type controls .

What are common technical challenges when working with C10orf71 antibodies?

Researchers working with C10orf71 antibodies may encounter several technical challenges due to the protein's intrinsic characteristics and expression patterns. One major challenge is the detection sensitivity, as C10orf71 is expressed specifically in cardiomyocytes but may not be as abundant as major structural proteins. Additionally, as an intrinsically disordered protein with an instability index of 65.67, C10orf71 may be prone to degradation during sample preparation, leading to inconsistent results. The high hydrophilicity of C10orf71 may also affect antibody binding efficiency in certain buffer conditions. When studying disease models with frameshift variants, researchers may encounter difficulty detecting truncated protein products, as demonstrated in studies where frameshift variant mRNA levels were significantly decreased due to nonsense-mediated mRNA decay. For immunofluorescence applications, the colocalization of C10orf71 with Z-disc and myofiber markers may present challenges in distinguishing specific signals from the complex structural background of cardiomyocytes. Finally, the limited commercial availability of validated C10orf71 antibodies targeting different epitopes restricts options for confirming results with multiple antibodies – a best practice in protein research. To address these challenges, researchers should optimize extraction protocols to minimize protein degradation, include appropriate controls, and potentially develop custom antibodies targeting multiple epitopes of C10orf71 .

How can C10orf71 antibodies be used to study protein-protein interactions in the context of heart disease?

C10orf71 antibodies can be instrumental in studying protein-protein interactions relevant to heart disease through multiple methodological approaches. Co-immunoprecipitation experiments using C10orf71 antibodies can pull down protein complexes from cardiac tissue or cardiomyocyte cultures, followed by mass spectrometry to identify interaction partners. This approach is particularly valuable for comparing interaction profiles between healthy samples and those from dilated cardiomyopathy patients or disease models. Proximity ligation assays (PLA) using C10orf71 antibodies in combination with antibodies against suspected interaction partners can visualize and quantify specific interactions within their native cellular context, allowing researchers to determine if these interactions are altered in disease states. Given that C10orf71-null cardiomyocytes exhibit impaired contractile function without affecting sarcomere structure, investigating interactions with components of the excitation-contraction coupling machinery would be especially relevant. Researchers can also use C10orf71 antibodies in ChIP-seq or RIP-seq experiments to explore potential roles in transcriptional or post-transcriptional regulation, as knockout studies have shown altered expression and splicing of contractile cardiac genes in C10orf71-KO mice. For advanced studies, C10orf71 antibodies can be used to visualize dynamic changes in protein interactions during cardiac stress or drug treatment, potentially identifying therapeutic targets for dilated cardiomyopathy caused by C10orf71 mutations .

What approaches can be used to detect post-translational modifications of C10orf71?

Detecting post-translational modifications (PTMs) of C10orf71 requires specialized approaches due to the protein's intrinsically disordered nature and specific expression pattern. Researchers should first employ phospho-specific antibodies if available, or general phospho-detection methods (such as ProQ Diamond staining) followed by Western blotting with C10orf71 antibodies to identify phosphorylated forms of the protein. Immunoprecipitation using C10orf71 antibodies followed by mass spectrometry analysis represents the most comprehensive approach to map the PTM landscape of C10orf71, including phosphorylation, acetylation, ubiquitination, or SUMOylation. For site-specific PTM analysis, targeted mass spectrometry methods such as selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) can be employed. To study the functional significance of identified PTMs, researchers can examine their changes during cardiac development (as C10orf71 expression increases significantly during heart development) or in response to pathological stress. Comparing PTM profiles between healthy hearts and those with dilated cardiomyopathy may reveal disease-relevant modifications. Since C10orf71 is an intrinsically disordered protein, PTMs may play particularly important roles in regulating its function or interactions, as is common for this class of proteins. Computational prediction tools can help prioritize potential PTM sites for experimental validation, focusing on conserved regions that might be functionally significant .

How can C10orf71 antibodies be integrated into high-throughput screening approaches?

C10orf71 antibodies can be strategically integrated into high-throughput screening approaches to accelerate discoveries related to dilated cardiomyopathy and cardiac function. One approach is to develop cell-based assays using cardiomyocytes (either primary cells or hiPSC-derived) with fluorescently-tagged C10orf71 antibodies to monitor protein localization, expression levels, or interactions in response to compound libraries. These screening platforms can identify small molecules that restore normal C10orf71 function or localization in cells modeling disease-causing mutations. Alternatively, researchers can develop ELISA-based high-throughput screens using C10orf71 antibodies to identify compounds that modulate C10orf71 protein levels or stability. For functional screens, C10orf71 antibodies can be used in combination with contractility assays in cardiomyocyte systems, similar to the approach used in rescue studies with omecamtiv mecarbil, which restored contractile function in C10orf71-null models. Pooled CRISPR screens targeting potential C10orf71 interaction partners, followed by immunofluorescence or Western blotting with C10orf71 antibodies, can help map the functional interaction network of this protein. Additionally, high-content imaging platforms using C10orf71 antibodies in conjunction with other cardiac markers can screen for compounds that normalize cardiac phenotypes in disease models. These high-throughput approaches can accelerate therapeutic discovery for dilated cardiomyopathy linked to C10orf71 dysfunction, potentially identifying cardiac myosin activators or other compounds that compensate for C10orf71 loss .

What statistical approaches are recommended for analyzing C10orf71 antibody data in comparative studies?

For robust analysis of C10orf71 antibody data in comparative studies, researchers should implement appropriate statistical approaches tailored to the experimental design and data characteristics. For Western blotting quantification, paired t-tests or Wilcoxon signed-rank tests are appropriate for comparing C10orf71 levels between two matched groups (e.g., diseased vs. healthy tissue from the same patients). For multiple group comparisons, ANOVA or Kruskal-Wallis tests followed by appropriate post-hoc tests should be employed. Given the high variability often observed in antibody-based quantification, power analyses should be conducted a priori to determine adequate sample sizes. For immunofluorescence quantification, mixed-effects models can account for the hierarchical nature of the data (multiple cells within tissues from multiple subjects). When analyzing correlations between C10orf71 levels and functional parameters (e.g., contractility measurements), multivariate regression models should be used to control for confounding variables such as age, sex, and comorbidities. For longitudinal studies examining C10orf71 expression over time, repeated measures ANOVA or linear mixed models are recommended. To address the specific expression pattern of C10orf71 in cardiomyocytes, consider using cardiomyocyte-specific normalization factors rather than global housekeeping genes. For all analyses, appropriate multiple testing correction should be applied when examining C10orf71 across different experimental conditions or in relation to multiple outcome measures .