zcrb1 Antibody

What is ZCRB1 and what cellular functions does it participate in?

ZCRB1 is an RNA-binding protein (RBP) encoded by the human ZCRB1 gene located on chromosome 12q12 that plays crucial roles in posttranscriptional regulation in cells . The protein contains zinc finger CCHC-type domains that facilitate specific interactions with RNA molecules, allowing it to participate in RNA processing mechanisms and splicing regulation . Research has demonstrated that ZCRB1 can specifically bind to the 5'-UTR of SARS-CoV RNA and promote viral RNA synthesis, suggesting a role in viral replication mechanisms . Recent studies have also implicated ZCRB1 in developmental processes, Wnt signaling pathways, and the regulation of m6A RNA methylation, highlighting its multifaceted roles in cellular biology . ZCRB1 appears to function within the U11/U12 minor spliceosome, a specialized complex responsible for processing a rare class of introns, indicating its specialized contribution to RNA processing machinery .

What experimental applications have been validated for ZCRB1 antibodies?

ZCRB1 antibodies have been successfully employed in Western blot assays across multiple cell lines including ARPE-19, Neuro-2a, Raji, and SMMC-7721 cells, as well as in mouse and rat testis tissues . Immunoprecipitation (IP) assays using ZCRB1 antibodies have proven effective for RNA-binding protein immunoprecipitation (RIP) experiments to identify RNA targets bound by ZCRB1 in cellular contexts . Researchers have utilized ZCRB1 antibodies for detecting protein expression differences between normal and disease states, particularly in cancer studies focusing on glioblastoma multiforme and lung adenocarcinoma . The antibodies have also been applied in detecting genetic alterations affecting ZCRB1 protein levels, as demonstrated in CRISPR/Cas9-edited cell lines where heterozygous mutations reduced ZCRB1 protein expression by approximately 30-50% . Though not explicitly stated in all research contexts, ZCRB1 antibodies likely function in immunofluorescence assays to determine subcellular localization patterns, given their use in related protein detection methods.

What are the recommended protocols for validating ZCRB1 antibody specificity?

Researchers should perform Western blot analysis using positive control samples known to express ZCRB1, such as ARPE-19 cells, Neuro-2a cells, Raji cells, SMMC-7721 cells, or testis tissue from rodent models, which have all been validated to express detectable levels of the protein . A genetic approach to validation involves creating CRISPR/Cas9-edited cell lines with reduced ZCRB1 expression and confirming corresponding reductions in antibody signal through both Western blotting and qRT-PCR, as demonstrated in studies using HEK293 cell models . Specificity testing should include negative controls such as competing peptide blocking assays or examination of tissues known to have minimal ZCRB1 expression to confirm the absence of non-specific binding . When conducting immunoprecipitation experiments, researchers should compare results with appropriate isotype control antibodies (e.g., mouse IgG) to distinguish specific from non-specific interactions, following established protocols from previous studies . Additional validation can include correlation analysis between protein detection (via antibody) and mRNA expression (via qRT-PCR) across multiple samples to confirm that the antibody signal accurately reflects gene expression patterns.

How should researchers optimize Western blot protocols for ZCRB1 detection?

Optimal protein extraction for ZCRB1 detection requires RIPA lysis buffer, which effectively solubilizes this nuclear protein while preserving its immunoreactivity for downstream antibody detection . Researchers should separate proteins using SDS-PAGE gels with appropriate percentage (typically 10-12% for ZCRB1's approximately 40 kDa size) followed by efficient transfer to PVDF membranes using standard electrophoretic transfer systems . For primary antibody incubation, the published protocol recommends overnight incubation at 4°C after blocking membranes for 2 hours at room temperature, which provides optimal signal-to-noise ratio for ZCRB1 detection . Signal development should utilize chemiluminescent detection systems such as the BeyoECL Star Kit, with image capture performed on systems like the MicroChemi chemiluminescent imaging system, followed by band intensity quantification using ImageJ software with β-actin as an appropriate endogenous control . When working with different tissue types, researchers may need to adjust lysis conditions or detergent concentrations to account for varying protein extraction efficiencies, though specific modifications for challenging tissues are not detailed in the available literature.

What experimental approaches are recommended for studying ZCRB1's RNA-binding capabilities?

RNA Immunoprecipitation (RIP) assays represent a primary method for investigating ZCRB1-RNA interactions, with established protocols utilizing the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit . This technique involves lysing approximately 2 × 10^7 cells and incubating the lysate with magnetic beads coupled with 5 μg anti-ZCRB1 antibody overnight at 4°C, with mouse IgG antibody serving as a negative control . Following immunoprecipitation, RNA bound to ZCRB1 can be isolated using proteinase K treatment and analyzed by qRT-PCR to identify specific RNA targets . Nascent RNA capture assays provide another valuable approach for investigating ZCRB1's role in RNA processing, involving incubation of cells with 0.5 mM 5-ethynyl uridine for 15 minutes to label newly synthesized RNAs, followed by biotinylation through Click chemistry and capture using streptavidin magnetic beads . For comprehensive identification of ZCRB1-bound RNAs, researchers can combine RIP with high-throughput sequencing (RIP-seq) or use CLIP (Crosslinking and Immunoprecipitation) techniques, though detailed protocols for these advanced methods were not specified in the provided references.

How can researchers effectively investigate ZCRB1's role in m6A RNA methylation pathways?

Researchers should first characterize ZCRB1's expression patterns in relation to known m6A methylation regulators through correlation analysis across multiple datasets, as demonstrated in studies of lung adenocarcinoma where ZCRB1 was identified as part of an m6A-related gene signature . RNA sequencing combined with differential gene expression analysis can help identify downstream targets affected by ZCRB1 modulation, as seen in studies where ZCRB1 heterozygous mutation resulted in dysregulation of over 4,000 protein-coding genes and 377 non-coding RNAs . Implementation of GAGE (Generally Applicable Gene-set Enrichment) analysis helps identify biological pathways significantly associated with ZCRB1 function, revealing its involvement in RNA metabolism processes and potential regulatory effects on cell signaling pathways . For mechanistic studies, researchers should consider creating cell models with modulated ZCRB1 expression (through overexpression constructs or CRISPR/Cas9 editing) followed by methylated RNA immunoprecipitation sequencing (MeRIP-seq) to directly assess changes in m6A modification patterns . Integration of experimental data with patient survival information from databases like TCGA enables researchers to contextualize findings within clinical frameworks, as illustrated by studies correlating ZCRB1 expression with prognosis in lung adenocarcinoma patients .

What techniques are most effective for studying ZCRB1 interactions with other proteins?

Co-immunoprecipitation (Co-IP) assays represent the gold standard for investigating protein-protein interactions involving ZCRB1, with published studies successfully using this approach to identify binding partners . Mass spectrometry following immunoprecipitation provides a powerful unbiased approach for identifying novel ZCRB1-interacting proteins, as demonstrated in studies validating encoded proteins and their interacting partners in the context of circRNA research . The GST pull-down assay offers another valuable in vitro method for confirming direct protein-protein interactions, utilizing GST-tagged ZCRB1 or its binding partners, with detection facilitated by antibodies against specific tags like GST or FLAG . For studying dynamic interactions in living cells, researchers can employ proximity ligation assays (PLA) or fluorescence resonance energy transfer (FRET) techniques, though specific protocols for these approaches with ZCRB1 were not detailed in the provided references. Validation of identified interactions should combine multiple methodologies and include appropriate controls such as immunoprecipitation with isotype-matched control antibodies and verification through bidirectional pull-down experiments.

How should researchers interpret conflicting results between ZCRB1 protein and mRNA expression studies?

Researchers encountering discrepancies between ZCRB1 protein and mRNA levels should first verify the specificity of antibodies used for protein detection through multiple validation methods, including testing in CRISPR/Cas9-edited cells with confirmed reductions in ZCRB1 expression . Post-transcriptional regulation mechanisms, such as microRNA targeting or RNA-binding protein interactions, may explain observed discrepancies, as ZCRB1 itself functions as an RNA-binding protein and could be subject to similar regulatory networks . Protein stability differences across experimental conditions or tissue types might contribute to inconsistent results, necessitating proteasome inhibitor experiments or cycloheximide chase assays to evaluate ZCRB1 protein half-life in different contexts . Technical factors including differences in sample preparation, normalization methods, or detection sensitivities between protein and RNA quantification techniques should be systematically evaluated and standardized across experiments . When analyzing patient-derived samples, researchers should consider potential tumor heterogeneity or microenvironmental influences that might differentially affect ZCRB1 mRNA and protein expression, requiring microdissection techniques or single-cell analysis approaches for more accurate interpretation .

What explains the tissue-dependent expression patterns of ZCRB1 observed across different cancer types?

The tissue-specific regulatory networks governing ZCRB1 expression likely contribute to its variable patterns across cancer types, with studies indicating differential expression in lymphoma (overexpression) versus lung adenocarcinoma where its expression pattern correlates with patient outcomes . Genetic alterations affecting the ZCRB1 locus may drive expression differences, with research identifying various mutation types including amplification, missense mutations, and deep deletions across cancer types, though ZCRB1 possessed relatively low alteration rates (approximately 3% in lung adenocarcinoma) . Epigenetic modifications, including DNA methylation and histone modifications at the ZCRB1 promoter region, likely play crucial roles in tissue-specific expression patterns, though specific epigenetic regulation mechanisms were not detailed in the provided references . The functional requirements for ZCRB1 may vary by tissue type based on the cell-specific demands for RNA processing, with certain tissues potentially requiring higher levels of minor spliceosome activity where ZCRB1 functions . Cancer progression stages could also influence ZCRB1 expression patterns, with some studies suggesting its role as an early biomarker in lung adenocarcinoma, potentially explaining why expression patterns differ between cancer types and progression states .

How can researchers accurately analyze ZCRB1's prognostic significance in cancer studies?

Multivariate Cox regression analysis represents an essential methodology for evaluating ZCRB1's independent prognostic value while accounting for clinical covariates like age, gender, stage, and existing biomarkers, as applied in studies of lung adenocarcinoma . Researchers should employ Kaplan-Meier survival curves with log-rank tests to visualize and statistically assess survival differences between patient groups stratified by ZCRB1 expression levels, using median expression as a logical cutoff point for high versus low expression categorization . Risk score modeling incorporating ZCRB1 alongside other molecular markers provides more robust prognostic information than single-gene analysis, as demonstrated in studies combining ZCRB1 with ADH1C and YTHDC2 to create a three-gene signature for lung adenocarcinoma prognosis prediction . Validation across multiple independent cohorts is crucial for establishing reliable prognostic markers, as exemplified by studies confirming ZCRB1's association with patient outcomes across three datasets (GSE31210, GSE50081, and TCGA) . Time-dependent ROC (Receiver Operating Characteristic) curve analysis should be utilized to assess the predictive accuracy of ZCRB1-based prognostic models at different time points (e.g., 3-year and 5-year survival), though specific ROC values were not provided in the available literature.

What statistical approaches are most appropriate for analyzing differential ZCRB1 expression in experimental models?

Quantitative RT-PCR data for ZCRB1 should be analyzed using the 2^-ΔΔCt relative quantification method with appropriate endogenous controls such as β-actin, ensuring consistent reference gene expression across experimental conditions . When analyzing RNA sequencing data, researchers should employ robust statistical packages that control for heteroscedasticity, as demonstrated in studies comparing ZCRB1 mutant clones to wild-type cells, which revealed significant dysregulation of thousands of genes . For protein-level quantification via Western blot, band intensities should be measured using software like ImageJ with normalization to loading controls such as β-actin, followed by appropriate statistical tests like t-tests for pairwise comparisons or ANOVA for multiple group analyses . Gene set enrichment analysis approaches, such as GAGE (Generally Applicable Gene-set Enrichment), provide valuable insights into biological pathways affected by ZCRB1 modulation, allowing researchers to contextualize expression changes within broader cellular processes . When comparing experimental models to patient data, researchers should consider batch effect correction and data normalization techniques to ensure comparability across different data sources and platforms .

How can researchers investigate ZCRB1's role in RNA splicing mechanisms?

Researchers should employ minigene splicing assays to directly assess ZCRB1's impact on splicing efficiency of target genes, particularly those containing U12-type introns, given ZCRB1's reported role in the U11/U12 minor spliceosome . RNA-seq with specific analysis pipelines designed to detect alternative splicing events (such as differential exon usage, intron retention, and splice junction analysis) provides a genome-wide view of splicing changes following ZCRB1 modulation . For mechanistic studies, CLIP-seq (Crosslinking and Immunoprecipitation followed by sequencing) can map ZCRB1-RNA binding sites with nucleotide resolution, revealing direct splicing targets and consensus binding motifs, though detailed protocols were not provided in the available references . Functional validation of identified splicing events should combine RT-PCR verification of specific splice junctions with minigene constructs containing wild-type and mutated ZCRB1 binding sites to confirm direct causality in splicing regulation . Co-IP studies examining ZCRB1's interactions with other splicing factors would further elucidate its position within splicing complex assembly and function, potentially revealing novel regulatory mechanisms in RNA processing pathways .

What approaches can researchers use to investigate ZCRB1's potential role in Wnt signaling?

Researchers investigating ZCRB1's role in Wnt signaling should quantify expression levels of key Wnt pathway components (such as Wnt7a, Wnt9B, Wnt11, Axin1, and Axin2) in response to ZCRB1 modulation, as studies have demonstrated significant upregulation of these genes in ZCRB1-deficient cells . Western blot analysis of phosphorylated LRP6, a downstream Wnt target, provides direct evidence of pathway activation status in response to ZCRB1 manipulation, with research showing increased phospho-LRP6 levels in ZCRB1 mutant cells . Functional Wnt signaling assays, such as TOPFlash/FOPFlash luciferase reporter systems measuring β-catenin-mediated transcriptional activity, would provide quantitative assessment of canonical Wnt pathway activity in response to ZCRB1 knockdown or overexpression . For mechanistic insights, researchers should investigate whether ZCRB1 regulates Wnt signaling through direct RNA-binding interactions with Wnt pathway component transcripts or through indirect mechanisms involving alternative splicing of Wnt regulators . Animal models with tissue-specific ZCRB1 modulation would enable in vivo validation of Wnt signaling effects, particularly in developmental contexts where both ZCRB1 and Wnt signaling play crucial roles, as suggested by zebrafish studies .

How can ZCRB1 antibodies be used to investigate its role in cancer progression?

Tissue microarray (TMA) analysis using ZCRB1 antibodies enables researchers to evaluate protein expression across large patient cohorts, correlating expression patterns with clinicopathological features and survival outcomes as demonstrated in lung adenocarcinoma studies . Immunohistochemistry in patient-derived xenograft (PDX) models allows for dynamic assessment of ZCRB1 expression changes during tumor progression and in response to therapeutic interventions, providing insights into its potential role as a biomarker or therapeutic target . Co-immunofluorescence studies examining ZCRB1 localization alongside markers of cancer stem cells, proliferation, or invasion can reveal associations with specific cancer phenotypes, though detailed protocols for such applications were not specified in the provided references . For functional studies, researchers should combine antibody-based detection methods with ZCRB1 knockdown or overexpression approaches in cancer cell lines, assessing effects on hallmark cancer phenotypes including proliferation, migration, invasion, and metabolic reprogramming . Integration of ZCRB1 protein expression data with multi-omics analyses (genomics, transcriptomics, proteomics) can help position ZCRB1 within broader cancer-related molecular networks, as suggested by studies linking it to pathways involving m6A RNA methylation in lung adenocarcinoma .

What are the best experimental approaches for studying ZCRB1's involvement in viral RNA interactions?

Researchers investigating ZCRB1's role in viral RNA interactions should perform RNA immunoprecipitation (RIP) assays using ZCRB1 antibodies followed by RT-PCR or sequencing to identify viral RNA sequences directly bound by ZCRB1, similar to studies demonstrating its binding to the 5'-UTR of SARS-CoV RNA . In vitro binding assays utilizing purified recombinant ZCRB1 protein and synthetic viral RNA fragments can determine binding affinities and sequence specificities, providing quantitative biochemical data on these interactions . Cell-based viral replication assays comparing infection outcomes in control versus ZCRB1-knockdown or ZCRB1-overexpressing cells can establish the functional significance of ZCRB1-viral RNA interactions on viral life cycles . For mechanistic insights, researchers should investigate whether ZCRB1 affects viral RNA stability, translation efficiency, or recruitment to cellular machinery, using techniques such as polysome profiling, RNA stability assays, and subcellular fractionation followed by ZCRB1 and viral RNA detection . Structural studies employing techniques like SHAPE (Selective 2'-hydroxyl acylation analyzed by primer extension) could map ZCRB1 binding sites on viral RNA secondary structures, providing insights into the molecular basis of these interactions.

What cell lines and tissues have been validated for ZCRB1 antibody applications?

The following table summarizes validated cell lines and tissues for ZCRB1 antibody detection:

This comprehensive validation across multiple species and tissue types demonstrates the versatility of ZCRB1 antibodies for diverse research applications. The confirmed reactivity in both human and rodent samples facilitates translational research across model systems. Researchers should note that while these samples have demonstrated positive detection, optimal antibody dilutions and protocol modifications may be necessary when adapting to new experimental systems or applications beyond Western blotting.

How does ZCRB1 expression correlate with patient survival in cancer studies?

Analysis of ZCRB1 expression in relation to patient outcomes reveals significant prognostic implications, particularly in lung adenocarcinoma:

The data consistently indicates that high ZCRB1 expression correlates with poorer outcomes in lung adenocarcinoma patients across multiple independent cohorts. When combined with other markers (ADH1C and YTHDC2) in a three-gene signature, the prognostic power is enhanced, suggesting potential utility as a biomarker panel. These findings contrast with ZCRB1's reported low expression in glioblastoma tissues, highlighting the context-dependent nature of its function across different cancer types and emphasizing the need for cancer-specific evaluation of its prognostic significance.

What genetic alterations affect ZCRB1 expression and function?

Studies investigating genetic modifications of ZCRB1 have revealed several important insights:

These genetic studies demonstrate that complete knockout of ZCRB1 appears to be incompatible with cell viability, as researchers were only able to generate heterozygous mutants despite multiple attempts to create complete knockouts. The substantial gene expression changes (over 4,000 differentially expressed genes) resulting from partial ZCRB1 loss highlight its critical role in transcriptome regulation. The existence of naturally occurring genetic alterations in patient populations further supports ZCRB1's biological significance and potential role in disease pathogenesis.

What are common challenges in ZCRB1 immunoprecipitation experiments and how can they be addressed?

Researchers performing ZCRB1 immunoprecipitation often encounter insufficient protein recovery, which can be addressed by optimizing lysis conditions with buffers containing appropriate detergent concentrations (typically RIPA buffer) and ensuring complete cell disruption through thorough homogenization . Non-specific binding in RIP experiments may obscure true ZCRB1-RNA interactions, necessitating rigorous controls including isotype-matched antibodies (e.g., mouse IgG) and pre-clearing lysates with protein A/G beads prior to immunoprecipitation . RNA degradation during immunoprecipitation procedures can be minimized by working quickly at cold temperatures, using RNase inhibitors in all buffers, and practicing stringent RNase-free technique throughout the experimental workflow . Antibody cross-reactivity may lead to misleading results, requiring validation of antibody specificity through Western blot analysis of input samples alongside immunoprecipitated material, with known positive and negative control samples providing essential reference points . For challenging applications involving low abundance targets, researchers can increase starting material (the referenced protocol used 2 × 10^7 cells), extend incubation times, or consider more sensitive detection methods such as nested PCR or digital PCR for analyzing immunoprecipitated RNA .

How should researchers interpret negative results in ZCRB1 antibody applications?

When encountering negative results with ZCRB1 antibody detection, researchers should first verify antibody activity using positive control samples such as ARPE-19, Neuro-2a, Raji, or SMMC-7721 cells, which have been validated to express detectable ZCRB1 protein . Technical factors including inadequate protein extraction, inappropriate blocking conditions, or suboptimal antibody dilutions should be systematically evaluated and optimized based on established protocols, such as overnight primary antibody incubation at 4°C following 2-hour blocking at room temperature . The context-dependent expression of ZCRB1 across different tissues and experimental conditions may result in genuinely low expression levels below detection thresholds, requiring more sensitive detection methods or sample enrichment through immunoprecipitation prior to analysis . The potential influence of biological variables such as cell cycle phase, culture conditions, or treatment effects on ZCRB1 expression should be considered when interpreting negative results, with appropriate experimental controls addressing these variables . If negative results persist despite optimization, researchers should consider alternative detection methods such as mass spectrometry or targeted RNA analysis to indirectly confirm ZCRB1 status within their experimental system .

What quality control measures are essential when working with ZCRB1 antibodies?

Lot-to-lot consistency testing is essential when receiving new ZCRB1 antibody batches, involving side-by-side comparison with previously validated lots using consistent positive control samples such as ARPE-19 or Raji cells to ensure comparable sensitivity and specificity . Specificity validation through genetic approaches, such as comparing signal intensity between wild-type and ZCRB1-depleted samples (CRISPR/Cas9-edited cells or siRNA knockdown), provides definitive confirmation of antibody specificity, as demonstrated in studies with HEK293 cell models . Optimization of experimental conditions for each application (Western blot, immunoprecipitation, immunofluorescence) is necessary, with particular attention to antibody dilution, incubation time and temperature, and detection method sensitivity, following established protocols that recommend overnight primary antibody incubation at 4°C . Inclusion of appropriate technical controls in each experiment, such as loading controls (β-actin) for Western blots, isotype controls for immunoprecipitation, and secondary-only controls for immunofluorescence, enables proper interpretation of results across different experimental contexts . Storage and handling practices, including aliquoting antibodies to avoid freeze-thaw cycles and maintaining appropriate storage temperatures, help preserve antibody performance over time, though specific storage recommendations for ZCRB1 antibodies were not detailed in the provided references.