CDC27A Antibody

What is CDC27 and what cellular functions does it regulate?

CDC27 (Cell Division Cycle 27) is a component of the anaphase-promoting complex (APC), functioning as an E3 ubiquitin ligase that catalyzes ubiquitin-mediated proteasomal degradation of type B cell cycle proteins . This protein plays a critical role in stimulating cell cycle transition from the middle stage to the later stage . In humans, CDC27 is encoded by the CDC27 gene and may also be known by several alternative names including APC3, HNUC, ANAPC3, CDC27Hs, and D0S1430E . The protein has a calculated molecular weight of 91.867 kDa and an observed molecular weight of approximately 92 kDa in experimental settings . Beyond its canonical role in cell cycle regulation, recent research has implicated CDC27 in autoimmune diseases, particularly systemic lupus erythematosus (SLE), suggesting broader functional implications in immune regulation .

What are the common applications of anti-CDC27 antibodies in research?

Anti-CDC27 antibodies are utilized across multiple experimental applications in research settings. Based on product specifications from various suppliers, these antibodies are commonly employed in Western Blot (WB), immunohistochemistry (IHC), immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), flow cytometry (FCM), immunocytochemistry (ICC), and immunofluorescence (IF) techniques . The versatility of these applications enables researchers to investigate CDC27 expression, localization, and interactions in diverse experimental contexts. For instance, Western blotting provides quantitative assessment of CDC27 protein levels, while immunofluorescence facilitates visualization of its subcellular distribution. In clinical research, quantitative polymerase chain reaction (qPCR) has been employed to measure CDC27 expression in peripheral blood mononuclear cells (PBMCs) from patients with SLE . The widespread availability of CDC27 antibodies from multiple suppliers (385 products across 30 suppliers) underscores their importance in contemporary biomedical research .

What considerations are important when selecting anti-CDC27 antibodies for different applications?

Selection of appropriate anti-CDC27 antibodies requires consideration of several key factors depending on the intended application. Researchers should first evaluate antibody specificity, ensuring the selected product demonstrates minimal cross-reactivity with other proteins . The immunogen used for antibody production is another critical consideration; for instance, some products utilize E.coli-derived human CDC27 recombinant protein (Position: H609-F824) as the immunogen . Species reactivity must align with experimental models—available antibodies show reactivity to human, mouse, and rat CDC27 . The antibody format (monoclonal versus polyclonal) should be selected based on the specific application; polyclonal antibodies often provide greater sensitivity but potentially lower specificity compared to monoclonals. Additionally, consider whether conjugated antibodies (e.g., with biotin, Cy3, or Dylight488) might be advantageous for particular applications like flow cytometry or immunofluorescence . For quantitative applications, validated antibodies with demonstrated performance in detecting the expected molecular weight (approximately 92 kDa for CDC27) should be prioritized . Finally, researchers should review available validation data, including Western blot images and immunohistochemistry results, to ensure the antibody performs reliably in the intended application.

What methodological approaches are most effective for studying CDC27 in SLE pathogenesis?

Investigating CDC27's role in SLE pathogenesis requires multi-faceted methodological approaches. Genetic screening represents a powerful starting point, as demonstrated by studies combining whole exon sequencing with bioinformatic tools including common-specific analysis, pedigree variant annotation analysis and search tool (pVAAST), Exomiser (combining phenotype and protein-protein interaction analysis), and family-based gene burden (FARVAT) . This comprehensive genetic approach successfully identified CDC27 as a potential causative gene for SLE. For expression analysis, quantitative PCR (qPCR) of peripheral blood mononuclear cells (PBMCs) has proven effective in comparing CDC27 expression between familial SLE patients, sporadic lupus patients, and healthy controls . When designing such studies, researchers should include adequate sample sizes for statistical power and appropriate controls for comparative analysis. Correlation analyses between CDC27 expression and clinical parameters (including C-reactive protein, erythrocyte sedimentation rate, and complement levels) provide valuable insights into the relationship between gene expression and disease activity . Longitudinal studies examining CDC27 expression before and after immunotherapy offer additional perspectives on its potential role as a biomarker for treatment response . Functional studies exploring the mechanistic consequences of CDC27 dysregulation would complement these approaches, potentially utilizing cell culture models with CDC27 knockdown or overexpression to elucidate downstream effects on immune cell function.

How can researchers optimize Western blot protocols for CDC27 detection?

Optimizing Western blot protocols for CDC27 detection requires careful attention to several critical parameters. Sample preparation represents the initial consideration—for cellular samples, lysis buffers containing protease inhibitors are essential to prevent degradation of the 92 kDa CDC27 protein . Protein concentration should be standardized across samples, typically 20-50 μg total protein per lane. For gel electrophoresis, 8-10% polyacrylamide gels typically provide optimal resolution for proteins in the 90-100 kDa range like CDC27. Transfer conditions require careful optimization; semi-dry transfer systems at 15-20V for 30-45 minutes or wet transfer systems at 100V for 60-90 minutes are commonly effective. When selecting primary antibodies, those designated as "Picoband" or similar premium designations often provide superior quality, high affinity, and strong signals with minimal background . Dilution ratios typically range from 1:500 to 1:2000 for primary antibodies, though optimal concentration should be empirically determined. Incubation time and temperature affect sensitivity—overnight incubation at 4°C generally yields optimal results for anti-CDC27 antibodies. For detection systems, enhanced chemiluminescence (ECL) provides sufficient sensitivity for most applications, though fluorescent secondary antibodies may offer advantages for quantitative analysis. When analyzing results, researchers should confirm that the detected band appears at the expected molecular weight of approximately 92 kDa . Multiple technical replicates and appropriate loading controls (such as β-actin or GAPDH) should be included to ensure reproducibility and normalization.

What is the relationship between CDC27 expression and clinical parameters in SLE?

Research examining CDC27 expression in SLE patients has revealed significant correlations with several clinical parameters. CDC27 expression in peripheral blood mononuclear cells (PBMCs) demonstrates a negative correlation with inflammatory markers, including C-reactive protein (CRP) (r=-0.919, P<0.01) and erythrocyte sedimentation rate (r=-0.804, P<0.001) . Conversely, CDC27 expression positively correlates with complement components C3 (r=0.927, P<0.001) and C4 (r=0.962, P<0.001) . These strong correlation coefficients suggest that CDC27 expression closely tracks with disease activity in SLE patients. The direction of these correlations—negative with inflammatory markers and positive with complement levels—indicates that lower CDC27 expression associates with higher disease activity. This relationship is further supported by observations that CDC27 expression increases in SLE patients following immunotherapy that successfully reduces disease activity . Importantly, no significant correlations were observed between CDC27 expression and other clinical indicators, including antinuclear antibodies, double-stranded DNA, proteinuria, and hematuria . These findings collectively suggest that CDC27 expression specifically relates to certain aspects of SLE pathophysiology, particularly inflammatory processes and complement activation, rather than representing a non-specific marker of autoimmunity. Understanding these relationships provides valuable insights for researchers investigating CDC27 as a potential biomarker or therapeutic target in SLE.

How can researchers effectively validate the specificity of anti-CDC27 antibodies?

Validating anti-CDC27 antibody specificity requires a multi-modal approach combining several complementary techniques. Western blotting represents the foundation of specificity validation—a high-quality anti-CDC27 antibody should detect a single predominant band at approximately 92 kDa, corresponding to the calculated molecular weight of the protein . Researchers should test the antibody across multiple cell lines or tissue types with varying CDC27 expression levels to confirm signal correlation with expected expression patterns. Genetic approaches provide powerful validation tools: comparing antibody signals in wild-type cells versus CDC27 knockdown (siRNA or shRNA) or knockout (CRISPR-Cas9) models should demonstrate corresponding signal reduction or elimination in the genetically modified samples. Immunoprecipitation followed by mass spectrometry can confirm that the antibody pulls down CDC27 rather than cross-reactive proteins. For immunohistochemistry or immunofluorescence applications, blocking peptide experiments are particularly valuable—pre-incubation of the antibody with the immunizing peptide should substantially reduce or eliminate specific staining . Cross-reactivity testing against closely related proteins, particularly other anaphase-promoting complex components, helps establish specificity within protein families. Comparative validation using multiple anti-CDC27 antibodies targeting different epitopes provides additional confidence when consistent results are observed. Finally, researchers should verify species reactivity claims by testing the antibody in human, mouse, and rat samples when cross-species applicability is required .

What sample preparation methods yield optimal results for CDC27 detection in clinical samples?

Optimizing sample preparation for CDC27 detection in clinical specimens requires attention to several critical factors that influence protein integrity and detection sensitivity. For peripheral blood mononuclear cells (PBMCs), which have been successfully used in CDC27 expression studies related to SLE , isolation should be performed using density gradient centrifugation with Ficoll-Paque or similar media, followed by immediate processing or appropriate preservation. If immediate processing is not possible, cryopreservation in liquid nitrogen with 10% DMSO and 90% FBS maintains protein integrity. For protein extraction, lysis buffers containing protease inhibitors are essential to prevent CDC27 degradation; RIPA buffer supplemented with 1mM PMSF, 1μg/ml aprotinin, and 1μg/ml leupeptin has proven effective for many researchers. Protein concentration should be determined using bicinchoninic acid (BCA) or Bradford assays, with standardization to ensure equal loading across samples. For immunohistochemical applications in tissue samples, fixation protocols significantly impact CDC27 epitope accessibility—4% paraformaldehyde provides superior antigen preservation compared to formalin for many epitopes. Antigen retrieval methods should be optimized; heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) at 95-100°C for 20 minutes typically yields good results for CDC27 detection. For clinical studies comparing patient cohorts, standardized collection, processing, and storage protocols are essential to minimize technical variability that could confound biological differences in CDC27 expression. Researchers should also consider collecting matched samples for multiple analytical methods (e.g., protein and RNA extraction) to enable methodological triangulation.

How should researchers interpret CDC27 expression data in the context of SLE diagnosis?

Interpretation of CDC27 expression data for SLE diagnosis requires careful analytical approaches that consider both statistical significance and clinical relevance. Based on receiver operating characteristic (ROC) curve analysis, CDC27 demonstrates promising diagnostic potential with sensitivity of 82.30% and specificity of 94.40% for SLE detection . When analyzing CDC27 expression data, researchers should first establish appropriate reference ranges in healthy control populations, accounting for potential confounding variables such as age, sex, and ethnicity. For quantitative PCR measurements, normalization to multiple stable reference genes rather than a single housekeeping gene enhances reliability. Statistical analysis should incorporate appropriate tests for comparing expression between groups—non-parametric tests like Mann-Whitney U test are often suitable given the typically non-normal distribution of gene expression data. For diagnostic applications, researchers should determine optimal cutoff values that balance sensitivity and specificity while considering the clinical context; the high specificity of CDC27 (94.40%) suggests particular utility as a confirmatory rather than screening biomarker . Correlation analysis with established SLE markers and disease activity indices provides valuable context for interpretation—the strong correlations between CDC27 expression and both inflammatory markers (CRP, erythrocyte sedimentation rate) and complement levels (C3, C4) reinforce its biological relevance to SLE pathophysiology . Longitudinal data analysis is particularly valuable, as the observed upregulation of CDC27 following effective immunotherapy suggests potential utility as a treatment response marker . Researchers should also consider integrating CDC27 expression with other biomarkers in multivariate models, which may enhance diagnostic accuracy beyond single-marker approaches.

What statistical approaches are most appropriate for analyzing CDC27 gene burden in familial SLE studies?

Family-based gene burden analysis for CDC27 in SLE pedigrees requires specialized statistical approaches that account for genetic relatedness within families. The FARVAT (family-based rare variant association test) methodology has demonstrated particular utility in this context, as evidenced by its successful application in identifying CDC27 as a potential causative gene for SLE . When designing such analyses, researchers should implement multiple complementary statistical tests—the BURDEN, CALPHA, and SKATO analyses within the FARVAT framework provide different perspectives on genetic associations . The BURDEN test aggregates rare variants within genes to increase statistical power, CALPHA detects variants with mixed effect directions, and SKATO (sequence kernel association test optimized) offers advantages for detecting associations with rare variants that have varying effect sizes and directions. For CDC27 specifically, all three tests identified significant associations at the extremely significant level (false discovery rate <0.05) . When interpreting results, researchers should consider several key metrics reported in the FARVAT analysis: NSAMP (number of samples), NVARIANT (number of variants), MAC (minor allele count), and NIMP (number of imputed samples) . Multiple testing correction is essential when examining numerous genes—false discovery rate (FDR) or Bonferroni correction should be applied to control Type I error rates. Complementary approaches including common-specific analysis, pVAAST (pedigree variant annotation, analysis and search tool), and Exomiser (combining phenotype and protein-protein interaction analysis) provide methodological triangulation, strengthening confidence when concordant results emerge across multiple analytical frameworks . Finally, findings from statistical genetics should be validated through functional studies examining CDC27 expression and its correlation with disease phenotypes.

How can researchers effectively combine genetic and expression data to understand CDC27's role in autoimmunity?

Integrating genetic and expression data provides a powerful approach for elucidating CDC27's role in autoimmune pathogenesis, particularly SLE. Researchers should begin by identifying genetic variants within or near CDC27 through whole exome sequencing of affected families and sporadic cases . These variants should be characterized according to their predicted functional impact using tools like SIFT, PolyPhen-2, or CADD, with particular attention to missense, nonsense, splicing, and regulatory region variants. Expression quantitative trait loci (eQTL) analysis can then determine whether identified variants associate with altered CDC27 expression levels. Direct measurement of CDC27 mRNA and protein expression in patient samples, particularly PBMCs, provides crucial validation of predicted expression effects . Correlation analysis between CDC27 expression and disease activity markers offers insights into the clinical relevance of expression variations . Pathway analysis incorporating CDC27 and its interaction partners, particularly within the anaphase-promoting complex, helps contextualize findings within broader biological processes. For mechanistic understanding, in vitro studies using cell lines with CDC27 variants introduced through CRISPR-Cas9 editing can reveal functional consequences on cell cycle progression, immune cell activation, or cytokine production. Animal models carrying CDC27 variants observed in SLE patients would provide in vivo validation of pathogenic mechanisms. This integrated approach has successfully demonstrated that CDC27 expression is decreased in both familial and sporadic SLE patients compared to healthy controls, correlates with disease activity markers, and increases following effective treatment —findings that collectively support its potential role in SLE pathogenesis and as a biomarker for disease monitoring.