C15orf41 Antibody

What is C15orf41 and why is it important to study?

C15orf41 is a protein encoded by the C15ORF41 gene that has been identified as the second causative gene underlying Congenital Dyserythropoietic Anaemia type I (CDA-I), a rare hereditary blood disorder characterized by ineffective erythropoiesis. The protein forms a tight, near-stoichiometric complex with Codanin-1 in human cells, interacting specifically with the C-terminal region of Codanin-1 . This interaction is crucial for understanding the molecular pathogenesis of CDA-I.

C15orf41 has been predicted to belong to the PD-(D/E)XK nuclease superfamily, suggesting a potential role in DNA processing, although its exact function remains to be fully characterized. Mutations in the C15ORF41 gene, such as pL178Q and pY94C, have been identified in CDA-I patients, indicating the protein's critical role in normal erythropoiesis . Understanding C15orf41's function and interactions is essential for elucidating the molecular mechanisms underlying CDA-I and potentially developing targeted therapies.

Which types of C15orf41 antibodies are available for research applications?

Based on the available literature, researchers have successfully produced and utilized several types of C15orf41 antibodies. Sheep polyclonal antibodies against GST-C15orf41 have been generated following standard immunization protocols . Additionally, commercially available rabbit anti-C15orf41 antibodies, such as Atlas Antibodies HPA061023, have been used in various experimental applications .

The antibodies are produced using standard methods, including expressing recombinant C15orf41 protein (often tagged with GST) in bacterial systems, purifying the protein, and immunizing animals such as sheep or rabbits. The resulting antisera can be further purified using affinity chromatography. These antibodies have been validated for various applications including Western blotting (WB) and immunofluorescence (IF), with each application requiring specific validation steps to ensure specificity .

What are the common applications for C15orf41 antibodies in basic research?

C15orf41 antibodies are valuable tools for several basic research applications. Primarily, they are used for detecting and quantifying C15orf41 protein expression in various cell types and tissues. Western blotting with C15orf41 antibodies allows researchers to assess protein levels and compare expression between different experimental conditions, cell types, or patient samples .

Immunofluorescence microscopy using C15orf41 antibodies enables visualization of the protein's subcellular localization. This has revealed that C15orf41 is predominantly localized in the nucleus, with smaller amounts present in the cytoplasm, suggesting roles in both cellular compartments . Co-immunoprecipitation experiments utilizing C15orf41 antibodies have been instrumental in identifying and characterizing protein-protein interactions, such as the complex formation between C15orf41 and Codanin-1 . Additionally, these antibodies can be used in flow cytometry and immunohistochemistry to study C15orf41 expression in different cell populations and tissue samples.

How should C15orf41 antibody specificity be validated prior to experimental use?

Thorough validation of C15orf41 antibody specificity is crucial to ensure reliable experimental results. A recommended approach is to use C15orf41 overexpression cells as a positive control, as demonstrated in published research . This involves transfecting cells with a C15orf41 expression construct and confirming increased antibody signal compared to non-transfected controls.

Additional validation methods include using cells with C15orf41 knockdown or knockout as negative controls, which should show reduced or absent antibody signal. Testing the antibody in multiple applications (e.g., Western blotting, immunofluorescence) can provide complementary evidence of specificity. For Western blotting, the antibody should detect a protein of the expected molecular weight (approximately 37 kDa for C15orf41), while non-specific bands should be minimal or absent . Pre-absorption controls, where the antibody is pre-incubated with purified C15orf41 protein before use, can further confirm specificity as this should neutralize the antibody and eliminate specific signals.

What are the optimal protocols for C15orf41 antibody production for research purposes?

Based on published methodologies, efficient production of C15orf41 antibodies involves a systematic approach starting with recombinant protein expression. For GST-C15orf41 antibody production, the protein is expressed in bacteria using appropriate expression vectors (e.g., plasmid DU49143) . The purified recombinant protein is then emulsified in Freund's adjuvant prior to immunization of the host animal, typically sheep or rabbits.

A standard immunization protocol involves collecting pre-immune serum before the first injection, followed by multiple antigen injections at 28-day intervals. Blood samples should be collected seven days after the second, third, and fourth injections. Approximately 750 ml of blood per bleed can yield 250–350 ml of serum . The collected blood should be allowed to clot overnight at 4°C, followed by centrifugation at 1500× g for 60 minutes to separate serum. The antibody can be further purified using affinity chromatography techniques, including antigen-coupled columns. Quality control should include testing antibody specificity using Western blotting and immunofluorescence against cells with varying levels of C15orf41 expression.

How can subcellular fractionation enhance the detection of C15orf41 in different cellular compartments?

Subcellular fractionation is a powerful technique for investigating the distribution of C15orf41 between nuclear and cytoplasmic compartments. Research has shown that C15orf41 is predominantly nuclear with some cytoplasmic presence, making fractionation particularly valuable for studying its compartment-specific functions .

An effective protocol involves using commercial reagents such as NE-PER™ Nuclear and Cytoplasmic Extraction Reagents, followed by Western blot analysis of the separated fractions. Proper controls are essential: TATA Binding Protein (TBP) serves as a nuclear fraction marker, while α-TUBULIN verifies cytoplasmic fraction purity . Densitometric quantification of C15orf41 signal normalized to these compartment-specific markers allows accurate determination of relative protein distribution. This approach revealed that while C15orf41 is more abundant in the nucleus, a significant portion is also present in the cytoplasm, suggesting multiple functional roles depending on localization . Complementary immunofluorescence using nuclear markers such as DRAQ5 can visually confirm the fractionation results and provide additional insights into subnuclear distribution patterns.

What approaches are recommended for studying C15orf41-Codanin-1 protein complex using antibodies?

Investigating the C15orf41-Codanin-1 complex requires sophisticated immunological approaches beyond simple detection. Co-immunoprecipitation (Co-IP) using either C15orf41 or Codanin-1 antibodies is the primary method for confirming and characterizing this interaction. Research has demonstrated that C15orf41 forms a tight, near-stoichiometric complex with Codanin-1, specifically interacting with its C-terminal region .

For Co-IP experiments, cell lysates should be prepared under conditions that preserve protein-protein interactions, typically using mild detergents like NP-40 or Triton X-100. The lysate is then incubated with either C15orf41 or Codanin-1 antibodies coupled to agarose or magnetic beads. After washing to remove non-specifically bound proteins, the immunoprecipitated complexes are analyzed by Western blotting using antibodies against both proteins . Reciprocal Co-IPs (immunoprecipitating with one antibody and blotting with the other, then vice versa) provide stronger evidence of interaction.

Proximity ligation assays (PLA) can provide in situ visualization of the C15orf41-Codanin-1 interaction with spatial resolution. For structural studies of the complex, researchers can employ purified recombinant proteins (His6-tagged C15orf41 and His6-tagged Codanin-1-C) expressed in bacterial systems and purified using Ni²⁺-NTA agarose chromatography . The interaction dynamics can be further characterized using techniques like surface plasmon resonance or isothermal titration calorimetry.

How should researchers interpret changes in C15orf41 expression in relation to CDAN1 expression?

Interpreting changes in C15orf41 expression requires careful consideration of its relationship with CDAN1 (the gene encoding Codanin-1). Multiple studies have demonstrated a significant direct correlation between C15orf41 and CDAN1 expression levels across various cell types and tissues . This correlation suggests coordinated regulation and functional interdependence between these two proteins.

When analyzing expression data, researchers should perform correlation analyses using appropriate statistical tests such as Pearson correlation. Studies have shown correlation coefficients of r=0.62 (p=0.01) in primary cells and r=0.99 (p<0.0001) across different cell lines . A reduction in C15orf41 expression, as observed in some CDA-I patients carrying C15orf41 mutations, may be accompanied by corresponding changes in CDAN1 expression . This pattern suggests that mutations affecting one partner may influence the stability or expression of the other.

The expression correlation extends across different hematopoietic cell populations, as demonstrated by in silico analysis of public gene expression datasets . For comprehensive interpretation, researchers should normalize gene expression to appropriate housekeeping genes (e.g., β-actin) and examine expression patterns across multiple cell types, including erythroid and non-erythroid lineages. Changes in the C15orf41:CDAN1 expression ratio, rather than absolute levels alone, may provide insights into pathological mechanisms in CDA-I and other disorders involving these proteins.

How can researchers address non-specific binding when using C15orf41 antibodies?

Non-specific binding is a common challenge when working with antibodies, including those against C15orf41. To minimize this issue, researchers should implement several optimization strategies. First, thorough blocking is essential—using 5% non-fat dry milk or bovine serum albumin (BSA) in TBS-T (Tris-buffered saline with Tween-20) for Western blotting, or appropriate blocking sera for immunofluorescence applications.

Antibody concentration titration is critical for determining the optimal working dilution that maximizes specific signal while minimizing background. Published research has used C15orf41 antibody at 1:500 dilution for Western blotting applications . Including appropriate controls in every experiment is essential—positive controls using cells overexpressing C15orf41 and negative controls using non-expressing cells or tissues help distinguish true signal from background .

For particularly problematic non-specific binding, pre-absorption of the antibody with the immunizing peptide or recombinant protein can significantly reduce background. Additionally, adjusting washing conditions (increasing number of washes, wash buffer composition, or washing time) can help eliminate non-specific signals. For immunofluorescence applications, using confocal microscopy with appropriate co-staining markers (such as DRAQ5 for nuclear visualization) can help distinguish true nuclear C15orf41 localization from potential artifacts .

What methodological considerations are important when comparing C15orf41 expression in patient samples versus controls?

Analyzing C15orf41 expression in patient samples requires careful methodological consideration to ensure valid comparisons. Sample preparation standardization is crucial—peripheral blood leukocytes (PBLs) or reticulocytes should be isolated using consistent protocols, as C15orf41 expression has been shown to be comparable between these cell types . RNA extraction methods should be consistent, with RNA quality and integrity verified before proceeding to cDNA synthesis and quantitative PCR.

For qRT-PCR analysis, appropriate reference genes (such as β-actin) must be selected and validated for the specific tissue type being studied . The 2^(-ΔCt) method is commonly used for relative quantification, but researchers should consider the efficiency of PCR amplification for both target and reference genes. Statistical analysis should account for the typically non-normal distribution of gene expression data, with appropriate tests such as the Mann-Whitney test or Student's t-test depending on data characteristics .

When analyzing protein expression by Western blotting, equal protein loading should be verified using housekeeping proteins such as β-actin for total lysates, TBP for nuclear fractions, or α-tubulin for cytoplasmic fractions . Densitometric analysis should be performed using appropriate software (such as Quantity One) to obtain integrated optical density values, with normalization to loading controls . Researchers should be aware that mutations in C15orf41 may affect protein stability differently than mRNA levels, as demonstrated by the differential effects of Y94S and H230P mutations .

How should researchers interpret C15orf41 protein turnover data in experimental systems?

Interpreting C15orf41 protein turnover data requires consideration of its complex regulation and relationship with binding partners. Time-course experiments have revealed interesting dynamics: while C15orf41 mRNA levels may show gradual increase following transfection, protein levels often peak early (around 16 hours post-transfection) and then progressively decrease despite sustained transcript expression . This pattern suggests post-translational regulation mechanisms affecting protein stability.

To accurately assess protein turnover, researchers should conduct parallel analyses of both mRNA (by qRT-PCR) and protein (by Western blotting) at multiple time points. Normalization to appropriate controls is essential—β-actin for mRNA expression and protein loading, with additional controls for transfection efficiency when using overexpression systems . Quantification should include densitometric analysis of Western blot signals, with results presented as relative expression levels compared to controls.

The observed discrepancy between mRNA and protein levels highlights the importance of post-translational regulation and potentially protein degradation pathways. Researchers may need to investigate whether C15orf41 undergoes ubiquitination or other modifications targeting it for degradation, particularly when studying mutant variants. The protein's half-life may also be influenced by its interaction with Codanin-1, suggesting that co-expression studies might provide additional insights into stability regulation mechanisms .

What are the best practices for analyzing C15orf41 localization using immunofluorescence techniques?

Analyzing C15orf41 localization by immunofluorescence requires careful technique optimization to obtain reliable results. Cell fixation method selection is critical—paraformaldehyde (typically 4%) preserves protein epitopes while maintaining cellular architecture. Permeabilization conditions must be optimized to allow antibody access to intracellular C15orf41 while preserving subcellular structures.

Antibody validation is essential before proceeding with localization studies. The specificity of rabbit anti-C15orf41 antibodies should be verified using overexpression systems as positive controls and appropriate negative controls . Working with the recommended dilution (typically starting at 1:500) and overnight incubation at 4°C often yields optimal results. Co-staining with subcellular markers is crucial for accurate localization determination—DRAQ5 is an effective nuclear marker that can be used alongside C15orf41 antibodies .

Image acquisition should utilize confocal microscopy for superior resolution of subcellular structures. Z-stack imaging can provide three-dimensional information about C15orf41 distribution. When analyzing images, researchers should assess both nuclear and cytoplasmic signals, as C15orf41 has been shown to be present in both compartments, though predominantly nuclear . Quantitative analysis of signal intensity in different compartments can be performed using appropriate imaging software. Researchers should note that no co-localization of C15orf41 with nucleoli has been observed, helping distinguish its distribution pattern from other nuclear proteins .

How can C15orf41 antibodies be used to investigate disease-associated mutations?

C15orf41 antibodies are valuable tools for investigating the molecular consequences of disease-associated mutations. For functional characterization of mutations like Y94S and H230P found in CDA-I patients, researchers can utilize expression constructs containing these variants followed by both transcriptional and protein analysis .

Western blotting with C15orf41 antibodies allows assessment of how mutations affect protein expression, stability, and potential degradation. Studies have shown that different mutations can have distinct effects—while H230P mutation leads to markedly reduced mRNA expression, Y94S appears to maintain normal mRNA levels while potentially affecting protein function through other mechanisms . Immunofluorescence microscopy can reveal whether mutations alter the subcellular localization of C15orf41, potentially providing insights into pathogenic mechanisms.

Co-immunoprecipitation experiments using C15orf41 antibodies can determine if disease-associated mutations disrupt the interaction with Codanin-1 or other binding partners. This approach can directly link molecular consequences of mutations to disease pathogenesis. Additionally, chromatin immunoprecipitation (ChIP) assays using C15orf41 antibodies might help identify potential DNA targets, particularly relevant given its predicted nuclease activity . For comprehensive analysis, researchers should compare wild-type and mutant proteins across multiple experimental readouts, including expression patterns, localization, protein-protein interactions, and potential enzymatic activities.

What experimental designs are recommended for investigating C15orf41's role in erythropoiesis?

Investigating C15orf41's role in erythropoiesis requires multifaceted experimental approaches combining cellular models with antibody-based detection methods. Erythroid differentiation models using cell lines like K562 can be induced with hemin (50 μM) following 24 hours of starvation to mimic erythroid maturation . Researchers should monitor differentiation through flow cytometry analysis of erythroid markers such as CD71 (expressed on proerythroblasts) and CD235a (expressed on proerythroblasts and orthochromatic erythroblasts).

Creating stable cell lines with either wild-type or mutant C15orf41 allows for assessment of how the protein influences erythroid differentiation . C15orf41 expression and localization can be tracked throughout differentiation using specific antibodies in both Western blotting and immunofluorescence applications. Cell cycle analysis using propidium iodide staining and flow cytometry can reveal how C15orf41 affects cell proliferation during erythropoiesis .

For more physiologically relevant models, researchers should consider primary CD34+ hematopoietic stem cells induced to undergo erythroid differentiation. Throughout the differentiation process, samples should be collected at defined stages for analysis of C15orf41 expression, localization, and interaction with Codanin-1. Comparison between normal controls and cells from CDA-I patients or gene-edited cells carrying disease-associated mutations can provide insights into pathogenic mechanisms. Chromatin immunoprecipitation using C15orf41 antibodies may identify DNA regions bound by the protein during erythroid differentiation, potentially revealing its role in regulating gene expression or DNA processing.

How can researchers utilize C15orf41 antibodies to explore its potential nuclease activity?

The predicted PD-(D/E)XK nuclease domain in C15orf41 suggests potential enzymatic activity that researchers can investigate using specialized approaches. In vitro nuclease assays using purified recombinant wild-type and mutant C15orf41 proteins can be designed to test activity against various DNA substrates (single-stranded, double-stranded, or structural intermediates). The presence or absence of nuclease activity can be monitored by gel electrophoresis to detect substrate cleavage.

Immunoprecipitation using C15orf41 antibodies followed by nuclease activity assays can determine if the protein possesses intrinsic enzymatic activity or associates with other nucleases in cellular contexts. Researchers should test different reaction conditions (varying pH, divalent cation concentrations, and salt concentrations) to identify optimal parameters for potential enzymatic activity.

Chromatin immunoprecipitation sequencing (ChIP-seq) using C15orf41 antibodies can identify genomic regions bound by the protein, potentially revealing preferred DNA sequences or structures. This approach could be particularly informative when comparing normal and CDA-I patient cells or when analyzing cells at different stages of erythroid differentiation. For comprehensive investigation, researchers should examine how disease-associated mutations (such as pL178Q and pY94C) affect potential nuclease activity, as these mutations might disrupt enzymatic function without necessarily affecting protein expression or stability .

What methodological approaches are recommended for investigating the temporal dynamics of C15orf41-Codanin-1 interaction?

Understanding the temporal dynamics of the C15orf41-Codanin-1 interaction requires sophisticated methodological approaches beyond static detection methods. Time-course experiments tracking both proteins during cell cycle progression or cellular differentiation can reveal dynamic regulation patterns. For such studies, synchronizing cells (using methods such as double thymidine block or nocodazole treatment) followed by release and sampling at defined time points allows precise temporal analysis.

Co-immunoprecipitation at sequential time points using either C15orf41 or Codanin-1 antibodies, followed by Western blotting for both proteins, can reveal changes in complex formation over time . Parallel analysis of total protein levels and subcellular localization provides context for interpreting interaction dynamics. Fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) using tagged proteins can provide real-time visualization of the interaction in living cells.

For studying dynamics during erythroid differentiation, researchers should collect samples at defined differentiation stages from models like hemin-induced K562 cells or primary erythroid cultures . Analysis of both proteins' expression, localization, and interaction throughout differentiation can reveal stage-specific regulation patterns. Given the known interaction between Codanin-1 and the histone chaperone ASF1, three-way interaction studies including ASF1 might provide insights into how C15orf41 fits into larger regulatory complexes controlling chromatin replication during erythropoiesis . Researchers should include appropriate controls for antibody specificity and consider how disease-associated mutations affect not only static interactions but also the dynamic regulation of complex formation.

Reference values for C15orf41 subcellular distribution

Comparison of wild-type and mutant C15orf41 expression patterns

Correlation between C15orf41 and CDAN1 expression across different systems