CCDC91 Antibody

What is CCDC91 and what cellular functions does it perform?

CCDC91 (Coiled-Coil Domain Containing 91) is a protein that plays a crucial role in cellular transport mechanisms, particularly in elastin trafficking and Golgi apparatus function. Recent research has demonstrated that CCDC91 is essential for normal elastin transport, with mutations in this gene being associated with conditions like Autosomal Dominant Keratitis-Ichthyosis-Deafness syndrome (AKE) . Functional studies have revealed that knockdown or mutation of CCDC91 results in distended Golgi cisternae, cytoplasmic vesicle accumulation, and altered tropoelastin distribution . Additionally, a novel isoform of CCDC91 has been identified that functions as a non-coding RNA involved in regulating osteogenic genes, suggesting diverse roles for different CCDC91 variants .

What types of CCDC91 antibodies are available for research applications?

Several types of CCDC91 antibodies are available for research, varying in the amino acid regions they target, host species, clonality, and conjugation status. Polyclonal antibodies raised in rabbit are common, targeting various epitopes including amino acids 1-180, 215-264, 322-350, and 1-411 of the CCDC91 protein . These antibodies are available in unconjugated forms as well as conjugated to reporter molecules like HRP, FITC, and Biotin for different experimental applications . Most commercially available antibodies are developed using recombinant human CCDC91 protein fragments as immunogens, such as the 1-180AA region, and are typically purified using Protein G affinity chromatography .

What are the validated applications for CCDC91 antibodies?

CCDC91 antibodies have been validated for several research applications including ELISA, Western blotting (WB), and Immunohistochemistry (IHC) . Immunofluorescence analysis is another critical application, particularly for investigating CCDC91's role in Golgi structure and function as well as elastin transport . In immunofluorescence studies, CCDC91 antibodies have been successfully used in conjunction with Golgi markers like GM130 to assess structural impairments in the Golgi apparatus following CCDC91 knockdown or mutation . The recommended dilutions for IHC applications range from 1:20 to 1:200, though optimal concentrations should be determined empirically for each experimental setup .

How should I design experiments to study CCDC91 function using genetic manipulation approaches?

When designing experiments to study CCDC91 function, consider using complementary genetic manipulation approaches. For RNA interference, design multiple shRNA or siRNA sequences targeting different regions of CCDC91 mRNA (NM_018318.5) . For example, four distinct shRNA sequences can be cloned into lentiviral vectors for stable knockdown in cell lines such as human skin fibroblasts (HSF) . For CRISPR/Cas9-mediated knockout, design guide RNAs targeting specific exons (e.g., exon 11) using validated design tools like CRISPR design tool (http://crispr.mit.edu/)[2] . Clone these sequences into appropriate vectors such as pSpCas9-2A-Puro (PX459) using restriction enzymes like BbsI . Always validate knockdown or knockout efficiency using quantitative RT-PCR and western blot analysis, with GAPDH as a housekeeping control for normalization .

What protocol should I follow for immunofluorescence analysis of CCDC91 in cell cultures?

For immunofluorescence analysis of CCDC91 in cultured cells, first grow cells on glass coverslips until reaching appropriate confluence . Fix cells with 4% formaldehyde for 10 minutes at room temperature, then wash thoroughly with PBS . Block non-specific binding by incubating with 1% BSA for 30 minutes . Incubate cells with primary antibodies against human CCDC91 (e.g., sc-514452, Santa Cruz) and appropriate organelle markers such as GM130 (12480T, Cell Signaling Technology) for Golgi visualization overnight at 4°C . After washing, apply suitable secondary antibodies like anti-mouse IgG Alexa Fluor 488 and anti-rabbit IgG Alexa Fluor 568/594, depending on your experimental design and microscopy setup . Counterstain nuclei with DAPI and acquire images using confocal microscopy, such as laser scanning microscopy (LSM880, Carl Zeiss) . This approach enables visualization of CCDC91 localization and assessment of Golgi structure integrity .

How can I validate the specificity of CCDC91 antibodies for my research?

To validate CCDC91 antibody specificity, implement a multi-layered approach. First, perform western blot analysis comparing wild-type cells with those undergoing CCDC91 knockdown or knockout to confirm the antibody detects bands of expected molecular weight that decrease in intensity with reduced CCDC91 expression . Second, conduct immunofluorescence staining in parallel with knockdown/knockout controls to verify reduction in signal intensity correlates with genetic manipulation of CCDC91 . Third, perform pre-absorption tests by incubating the antibody with excess recombinant CCDC91 protein (e.g., the immunogen used for antibody production) before staining to confirm signal elimination . Fourth, use multiple antibodies targeting different epitopes of CCDC91 (e.g., AA 1-180, AA 215-264, AA 322-350) and compare their staining patterns for consistency . Finally, verify co-localization with known interacting partners or organelle markers such as GM130 for Golgi-associated CCDC91 .

How can I investigate the role of CCDC91 in elastin transport and processing?

To investigate CCDC91's role in elastin transport, implement a comprehensive approach combining genetic manipulation with protein localization studies. First, establish CCDC91 knockdown or knockout cell models using siRNA, shRNA, or CRISPR/Cas9 techniques in relevant cell types such as human skin fibroblasts (HSF) . Then, perform co-immunofluorescence studies using antibodies against CCDC91, tropoelastin (TE, e.g., PR398, Elastin Products Company), and Golgi markers (e.g., GM130) . This allows visualization of potential tropoelastin accumulation in the Golgi apparatus and abnormal extracellular aggregates in CCDC91-deficient cells . Additionally, examine human aortic alpha elastin (PR533, EPC) and human fibrillin-1 (NBP1-84722, NOVUS biologicals) distribution to assess potential effects on elastic fiber assembly and microfibril formation . For functional assessment, analyze lysyl oxidase activity, which is critical for elastin crosslinking, and evaluate changes in cellular morphology and organelle structure, particularly focusing on Golgi cisternae distension and cytoplasmic vesicle accumulation .

What approaches should I use to study mutations in the CCDC91 gene and their functional consequences?

To study CCDC91 mutations and their functional impact, begin with genomic analysis to identify mutations of interest, such as the splicing mutation (G>A) that causes exon 11 skipping and results in a 59-amino-acid deletion (L309-Q367del) . Recreate these mutations using CRISPR/Cas9 genome editing in relevant cell lines like HEK293T . Design guide RNAs targeting the specific exon (e.g., exon 11) and validate the edited cells by sequencing the CCDC91 cDNA to confirm the intended genetic modification . Alternatively, for novel isoforms, clone the full-length cDNA into expression vectors like pcDNA3.1(-) for overexpression studies . Evaluate functional consequences through multiple approaches: 1) Analyze Golgi structure using immunofluorescence with GM130 and CCDC91 antibodies; 2) Assess protein trafficking, particularly of elastin and associated proteins; 3) Examine cellular ultrastructure with transmission electron microscopy to detect distended Golgi cisternae and vesicle accumulation; and 4) Perform rescue experiments by expressing wild-type CCDC91 in mutant cells to confirm phenotype specificity .

How can I explore the relationship between CCDC91 and non-coding RNA functions?

To investigate CCDC91's role as a non-coding RNA regulator, begin by identifying any novel CCDC91 isoforms through RNA sequencing and isoform-specific PCR . For functional analysis of these isoforms, design siRNAs targeting the specific isoform of interest and transfect them using appropriate reagents like Lipofectamine RNAiMAX at optimized concentrations (e.g., 10 nM) . Validate knockdown efficiency using quantitative RT-PCR with isoform-specific primers . To study regulatory mechanisms, construct reporter plasmids containing the promoter region of the novel CCDC91 isoform (e.g., -337 to +356 of exon 1) cloned into luciferase reporter vectors like pGL4.10[luc2] . For microRNA interaction studies, identify potential microRNA response elements (MREs) within the CCDC91 transcript using prediction tools like RNA22 or miRDB . Clone three tandem repeats of wild-type or mutated MRE sequences into dual-luciferase reporter vectors like pmirGLO to validate direct interactions . Finally, identify downstream targets of CCDC91-regulated microRNAs using tools like miRDB, followed by validation with qPCR, western blotting, and functional assays specific to the predicted pathways, such as osteogenic differentiation if studying bone-related effects .

How should I troubleshoot inconsistent or weak signals when using CCDC91 antibodies?

When facing inconsistent or weak signals with CCDC91 antibodies, systematically troubleshoot by first optimizing antibody concentration, testing a range of dilutions (e.g., 1:20 to 1:200 for IHC applications) . Next, evaluate fixation protocols; over-fixation can mask epitopes while under-fixation may compromise cell morphology and protein retention . For immunofluorescence, extend primary antibody incubation time to overnight at 4°C and optimize antigen retrieval methods if applicable . Consider antibody selection factors: different CCDC91 antibodies target distinct epitopes (AA 1-180, AA 215-264, AA 322-350), and mutations or isoform expression may affect epitope availability . Test alternative antibodies targeting different regions of the protein, particularly if studying mutant CCDC91 with deletions (like L309-Q367del) . Protein expression levels vary across cell types; therefore, use positive control samples with known CCDC91 expression . Finally, for low abundance proteins, consider signal amplification techniques or more sensitive detection systems, and always run parallel experiments with knockdown/knockout controls to confirm signal specificity .

What bioinformatic approaches can I use to analyze CCDC91 function and its genetic variants?

For comprehensive bioinformatic analysis of CCDC91 function and variants, utilize a multi-pronged approach. To predict the effects of non-coding variants on gene expression, employ specialized tools like MENTR (mutation effect prediction on non-coding RNA transcription), using established thresholds (e.g., absolute mutation effect > 0.05) to predict expression changes . For microRNA target prediction within CCDC91 transcripts, utilize algorithms such as RNA22 and miRDB, and subsequently use miRDB to identify potential targets of these microRNAs . To analyze protein structure and functional domains, particularly for assessing the impact of mutations like the 59-amino-acid deletion (L309-Q367del), use protein structure prediction tools and analyze conservation patterns across species . For splice site variants, employ tools that predict alterations in splicing patterns and exon usage . Genome-wide association studies (GWAS) data can be analyzed to identify significant variants in CCDC91 associated with specific phenotypes, applying appropriate statistical thresholds (e.g., p < 0.05) to determine significance . Finally, perform pathway analysis to understand CCDC91's role in broader cellular networks, particularly in elastin transport and Golgi function .

How can I quantify and statistically analyze CCDC91 expression levels in experimental samples?

For robust quantification and statistical analysis of CCDC91 expression, implement a comprehensive approach. When using quantitative RT-PCR, design primers specific to CCDC91 (refer to validated primer sequences in literature) and normalize expression to stable reference genes like GAPDH . Perform experiments in biological triplicates and technical duplicates to ensure reproducibility . For western blot quantification, normalize CCDC91 band intensity to loading controls and measure using densitometry software, presenting results as relative fold change compared to controls . For immunofluorescence quantification, capture images using consistent exposure settings and analyze signal intensity using software like ImageJ, measuring parameters such as mean fluorescence intensity, area of staining, or colocalization coefficients with markers like GM130 . For statistical analysis, first assess data normality using Shapiro-Wilk or similar tests . For comparing two groups (e.g., control vs. knockdown), use appropriate tests such as Student's t-test for normally distributed data or Mann-Whitney U test for non-parametric data . For multiple group comparisons, employ ANOVA followed by post-hoc tests like Tukey's or Bonferroni . Consider p < 0.05 as statistically significant, and present data as mean ± standard deviation with individual data points visible when possible .

How can CCDC91 antibodies be used in studying disease mechanisms related to elastin dysfunction?

CCDC91 antibodies offer valuable tools for investigating disease mechanisms associated with elastin dysfunction, particularly conditions like Autosomal Dominant Keratitis-Ichthyosis-Deafness syndrome (AKE) . Begin by establishing disease models using patient-derived cells or by introducing disease-specific mutations (such as the G>A splicing mutation causing exon 11 skipping) in cell lines using CRISPR/Cas9 . Utilize CCDC91 antibodies in combination with tropoelastin (TE) antibodies to perform co-immunofluorescence studies, examining alterations in elastin trafficking, Golgi localization, and extracellular deposition patterns . Compare TE distribution patterns between wild-type and CCDC91-mutant cells to identify abnormal tropoelastin accumulation in the Golgi apparatus and formation of irregular extracellular aggregates . Additionally, assess changes in the Golgi apparatus structure using CCDC91 antibodies alongside Golgi markers like GM130, documenting distended Golgi cisternae and other morphological abnormalities . For comprehensive analysis, combine antibody-based visualization techniques with transmission electron microscopy to detect ultrastructural changes in vesicle trafficking pathways and with functional assays measuring elastin secretion, deposition, and cross-linking efficiency .

What is the recommended approach for studying interactions between CCDC91 and other proteins involved in cellular trafficking?

To study interactions between CCDC91 and other trafficking proteins, implement a systematic protein-protein interaction analysis strategy. Begin with co-immunoprecipitation (Co-IP) experiments using CCDC91 antibodies to pull down protein complexes, followed by mass spectrometry to identify novel interaction partners or western blotting to confirm suspected interactions . Validate these interactions through reciprocal Co-IP experiments and proximity ligation assays (PLA), which provide visual confirmation of protein proximity in situ . For spatial relationship analysis, perform multi-color immunofluorescence using CCDC91 antibodies alongside antibodies against trafficking machinery components, particularly those associated with the Golgi apparatus (e.g., GM130) and vesicular transport systems, analyzing colocalization using confocal microscopy and quantitative colocalization metrics . To assess functional relationships, conduct parallel knockdown experiments of CCDC91 and identified interaction partners, examining changes in protein localization, Golgi morphology, and cargo transport efficiency, particularly focusing on elastin trafficking . For real-time dynamics, consider live-cell imaging using fluorescently tagged CCDC91 and partner proteins to visualize trafficking events and protein complex formation in living cells, providing temporal information about interaction kinetics .

How can I integrate CCDC91 antibody-based techniques with other methodologies for comprehensive protein function studies?

For comprehensive CCDC91 functional studies, integrate antibody-based techniques with complementary methodologies in a multi-modal approach. Combine immunofluorescence microscopy using CCDC91 antibodies with live-cell imaging of fluorescently tagged cargo proteins like tropoelastin to track trafficking dynamics in real-time, correlating static antibody-based observations with dynamic cellular processes . Integrate biochemical fractionation techniques with western blotting using CCDC91 antibodies to analyze protein distribution across cellular compartments, particularly focusing on Golgi-associated fractions versus vesicular components . Complement genetic manipulation approaches (siRNA knockdown, CRISPR/Cas9 knockout) with rescue experiments using wild-type or mutant CCDC91 constructs, followed by antibody-based detection of phenotypic changes in Golgi structure and cargo distribution . Implement omics approaches, such as proteomics analysis of cells with altered CCDC91 expression, with validation of key findings using CCDC91 antibodies for western blotting or immunofluorescence . For transcriptional regulation studies, combine chromatin immunoprecipitation (ChIP) of transcription factors potentially regulating CCDC91 with expression analysis using CCDC91 antibodies . Finally, correlate in vitro findings with tissue-level analyses using immunohistochemistry with CCDC91 antibodies on relevant tissue sections, particularly those affected in elastin-related disorders, to bridge cellular mechanisms with tissue pathology .

What controls should be included when using CCDC91 antibodies in various applications?

When using CCDC91 antibodies, include a comprehensive set of controls to ensure result validity. For all applications, incorporate a negative control omitting the primary antibody while maintaining all other reagents and conditions to assess background and non-specific binding of secondary antibodies . Include positive control samples with known CCDC91 expression, such as specific cell lines or tissues documented to express the protein . For validation of antibody specificity, use CCDC91 knockdown or knockout samples created through siRNA, shRNA, or CRISPR/Cas9 approaches as biological negative controls . When performing immunofluorescence, include co-staining with established markers like GM130 for Golgi localization to confirm expected subcellular distribution patterns . For quantitative applications like western blotting or qPCR, include dilution series to confirm signal linearity and loading controls (GAPDH, β-actin) for normalization . When studying specific mutations or variants of CCDC91, include both wild-type and mutant samples processed identically to directly compare expression patterns and levels . For therapeutic or intervention studies, incorporate time-matched vehicle controls alongside treatment conditions to distinguish specific effects from experimental artifacts .

How should I select the most appropriate CCDC91 antibody for my specific research question?

Selection of the optimal CCDC91 antibody requires careful consideration of several factors aligned with your research objectives. First, identify your target epitope based on the specific CCDC91 region of interest; antibodies targeting different regions (AA 1-180, AA 215-264, AA 322-350, AA 1-411) may yield different results, particularly when studying mutations or splice variants that affect specific domains . For instance, when investigating mutations affecting exon 11 (L309-Q367del), antibodies targeting this region may show altered binding patterns . Second, consider the intended application; some antibodies are validated specifically for certain techniques like ELISA, western blotting, or immunohistochemistry . Third, evaluate species reactivity requirements; while many CCDC91 antibodies react with human proteins, cross-reactivity with mouse, rat, or other species varies significantly between antibodies . Fourth, assess clonality needs; polyclonal antibodies may provide stronger signals by recognizing multiple epitopes but might show batch-to-batch variation, while monoclonal antibodies offer higher specificity and consistency . Finally, consider conjugation requirements; select from unconjugated antibodies for flexible detection strategies or pre-conjugated options (HRP, FITC, Biotin) for specific detection systems, depending on your experimental design and available equipment .