C17orf58 Antibody

What is C17orf58 and why are antibodies against it useful in research?

C17orf58 (Chromosome 17 Open Reading Frame 58), also known as UPF0450 protein C17orf58, is a protein encoded by the C17orf58 gene in humans. This protein has been the subject of various research studies, though its complete function remains under investigation. Antibodies against C17orf58 provide valuable tools for detecting, localizing, and studying this protein within biological samples .

The utility of C17orf58 antibodies in research stems from their ability to specifically recognize and bind to C17orf58 protein in various experimental applications. This specificity allows researchers to determine protein expression patterns across different tissues, investigate protein-protein interactions, and analyze changes in protein levels under various experimental conditions. Additionally, these antibodies enable researchers to explore potential roles of C17orf58 in cellular processes and disease pathways .

When selecting a C17orf58 antibody for research, it's important to consider the specific application requirements, including the species reactivity, antibody type (monoclonal vs. polyclonal), and validated applications such as Western blotting, immunohistochemistry, or immunofluorescence.

What applications are C17orf58 antibodies validated for?

C17orf58 antibodies have been validated for multiple research applications, with the specific applications varying by antibody format and manufacturer. Based on the available data, these antibodies are primarily validated for the following techniques:

Western Blotting (WB): Mouse monoclonal C17orf58 antibody (OTI6E9) has been validated for Western blot applications at a dilution of 1/2000, with a predicted band size of 11 kDa. This antibody has been tested on HEK-293T cell lysates with overexpressed C17orf58 protein .

Immunohistochemistry (IHC): Rabbit polyclonal antibodies against C17orf58 are suitable for immunohistochemistry at dilutions ranging from 1:200 to 1:500. These antibodies have been extensively characterized through the Human Protein Atlas project, which includes testing on arrays of normal human tissues and common cancer types .

Immunofluorescence (IF): C17orf58 antibodies are applicable for immunofluorescence studies at concentrations of 0.25-2 μg/mL. This application allows for subcellular localization studies of the C17orf58 protein .

ELISA: Several C17orf58 antibody formats, including those conjugated with biotin, FITC, or HRP, are validated for ELISA applications, enabling quantitative detection of the protein in solution .

When designing experiments utilizing these applications, researchers should consider control samples, optimization of antibody concentrations, and appropriate detection methods to ensure reliable and reproducible results.

How should researchers optimize Western blot protocols when using C17orf58 antibodies?

Optimizing Western blot protocols for C17orf58 antibody detection requires careful consideration of several experimental parameters. Based on the validated protocols and technical information, researchers should follow these methodological steps:

Sample Preparation: Prepare protein lysates from appropriate cellular or tissue sources. For C17orf58 detection, HEK-293T cell lysates have been successfully used as positive controls, particularly those transfected with C17orf58 cDNA . Include both experimental samples and appropriate controls, such as untransfected cells or cells transfected with empty vector.

Protein Loading and Separation: Load approximately 5-20 μg of protein per lane for optimal detection. Given the relatively small size of C17orf58 (predicted band size of 11 kDa), use higher percentage (12-15%) SDS-PAGE gels for better resolution of low molecular weight proteins .

Transfer Conditions: Optimize transfer conditions for small proteins by using appropriate transfer buffers (containing methanol) and shorter transfer times to prevent small proteins from passing through the membrane.

Blocking and Incubation: Use 5% non-fat dry milk or BSA in TBST for blocking (1 hour at room temperature). Incubate with primary antibody overnight at 4°C with gentle agitation. Wash thoroughly with TBST before applying appropriate secondary antibody.

Detection Method: Choose an appropriate detection method based on the expected abundance of C17orf58 in your samples. Enhanced chemiluminescence (ECL) is suitable for most applications, but more sensitive detection systems may be required for low-abundance samples.

By meticulously optimizing these parameters, researchers can achieve specific and sensitive detection of C17orf58 protein while minimizing background noise and non-specific binding.

What species reactivity should be considered when selecting C17orf58 antibodies?

Species reactivity is a critical consideration when selecting C17orf58 antibodies for research applications. From the available information, most commercially available C17orf58 antibodies demonstrate the following reactivity profiles:

Human Reactivity: Most C17orf58 antibodies are primarily developed against and validated for human samples. Both the mouse monoclonal (OTI6E9) and rabbit polyclonal antibodies show confirmed reactivity with human C17orf58 protein . These antibodies recognize human C17orf58 in various applications including Western blotting, immunohistochemistry, and immunofluorescence.

When selecting an antibody for cross-species applications, researchers should:

• Compare the immunogen sequence used to generate the antibody with the target sequence in the species of interest

• Review any available cross-reactivity data from manufacturers

• Consider testing the antibody on positive control samples from the target species

• If no validated antibody exists for the species of interest, select antibodies raised against conserved epitopes of the protein

This thoughtful approach to species reactivity will help ensure successful experimental outcomes when working with C17orf58 across different model systems.

How can researchers validate the specificity of C17orf58 antibodies to avoid cross-reactivity issues?

Validating antibody specificity is critical for ensuring reliable research results, particularly for proteins like C17orf58 where limited characterization exists. Researchers should implement a comprehensive validation strategy that includes multiple approaches:

Genetic Validation: One of the most rigorous approaches involves using genetic manipulation techniques. Creating C17orf58 knockout (KO) cell lines via CRISPR-Cas9 provides an excellent negative control. The absence of signal in KO samples when probed with the C17orf58 antibody strongly supports specificity. Similarly, overexpression systems, such as HEK-293T cells transfected with C17orf58 cDNA (as shown in the Western blot data), provide valuable positive controls .

Immunoprecipitation-Mass Spectrometry (IP-MS): This technique can identify all proteins captured by the antibody. Perform immunoprecipitation with the C17orf58 antibody followed by mass spectrometry analysis of the precipitated proteins. High specificity is indicated when C17orf58 represents the predominant protein identified.

Peptide Competition Assays: Pre-incubating the antibody with excess immunizing peptide (such as the sequence FRVHMLALDSSSCNKPCPEFKPGSRYIVMGHIYHKRRQLPTALLQVLRGRLRPGDGLLRSSSSYVKRFNRKREGQIQGAIH for certain rabbit polyclonal antibodies) should abolish or significantly reduce specific signals in Western blot, IHC, or IF applications . Persistent signals after peptide competition suggest non-specific binding.

Multiple Antibody Concordance: Using multiple antibodies targeting different epitopes of C17orf58 and comparing their staining/detection patterns provides additional validation. Concordant results with antibodies recognizing different regions strongly support specificity for the target protein.

Correlation with mRNA Expression: Correlating protein detection levels with mRNA expression data across tissues or cell types can provide additional evidence for antibody specificity. Techniques like RT-qPCR or consulting RNA-seq databases can supply the mRNA expression data for comparison.

What are the methodological considerations for using C17orf58 antibodies in multiplexed immunofluorescence studies?

Multiplexed immunofluorescence allows simultaneous detection of multiple protein targets in a single sample, providing valuable spatial context and co-expression information. When incorporating C17orf58 antibodies into multiplexed studies, researchers should address several methodological considerations:

Antibody Selection and Characterization:
Select a C17orf58 antibody validated for immunofluorescence applications, such as the rabbit polyclonal antibody recommended at concentrations of 0.25-2 μg/mL . Prior to multiplexing, validate the antibody in single-plex experiments to determine optimal concentration, incubation conditions, and expected staining pattern. For advanced studies, consider pre-conjugated fluorescent C17orf58 antibodies or antibody fragments to minimize issues with secondary antibody cross-reactivity .

Species Compatibility and Panel Design:
When designing a multiplexed panel, select primary antibodies raised in different host species to enable the use of species-specific secondary antibodies. If this is not possible, consider directly conjugated primary antibodies or implement sequential staining protocols with appropriate blocking steps between rounds. Ensure that other antibodies in your panel have been validated for the same fixation and antigen retrieval conditions as required for C17orf58 detection.

Signal Separation and Spectral Unmixing:
Choose fluorophores with minimal spectral overlap to reduce bleed-through. For complex panels, consider using spectral unmixing algorithms during image analysis. Be mindful of the expected subcellular localization of C17orf58 and other target proteins to accurately interpret colocalization patterns. Include single-stained controls for each fluorophore to establish proper signal separation parameters.

Optimizing Antigen Retrieval and Fixation:
Different antibodies may require specific fixation and antigen retrieval methods. For C17orf58 detection in FFPE tissues, test different antigen retrieval methods (heat-induced epitope retrieval with citrate buffer at pH 6.0 or EDTA buffer at pH 9.0) to determine optimal conditions that maintain both C17orf58 immunoreactivity and tissue morphology while remaining compatible with other antibodies in the panel.

Controls and Quantification Strategies:
Include appropriate controls: unstained samples, single-color controls, isotype controls, and biological positive and negative controls. For quantitative analysis, develop consistent acquisition parameters and analysis workflows. Consider automated image analysis platforms for unbiased quantification of staining patterns, especially when evaluating potential colocalization of C17orf58 with other proteins of interest.

By meticulously addressing these methodological considerations, researchers can successfully incorporate C17orf58 antibodies into multiplexed immunofluorescence studies, yielding reliable and informative results about the spatial context of C17orf58 expression relative to other proteins.

How can researchers differentiate between isoforms of C17orf58 using antibody-based techniques?

Differentiating between protein isoforms represents a significant challenge in antibody-based detection methods. For C17orf58, which has been referred to with isoform designations such as "UPF0450 protein C17orf58 isoform a" , researchers should employ a strategic approach:

Epitope Mapping and Antibody Selection:
Begin by analyzing the sequence differences between C17orf58 isoforms through bioinformatics resources. Identify unique regions that distinguish each isoform. Select or develop antibodies targeting isoform-specific epitopes. Commercial antibodies should be evaluated for the specific immunogen sequence used in their production, such as the sequence FRVHMLALDSSSCNKPCPEFKPGSRYIVMGHIYHKRRQLPTALLQVLRGRLRPGDGLLRSSSSYVKRFNRKREGQIQGAIH referenced for certain polyclonal antibodies . Determine if this sequence is common to all isoforms or specific to particular variants.

High-Resolution Protein Separation:
Employ techniques that provide superior resolution of closely related protein variants. Use gradient gels (4-20%) for SDS-PAGE to maximize separation of isoforms with subtle size differences. Consider using Phos-tag™ acrylamide gels if isoforms differ in phosphorylation status. For even greater resolution, implement 2D electrophoresis (isoelectric focusing followed by SDS-PAGE) to separate isoforms based on both isoelectric point and molecular weight differences.

Isoform-Specific Knockdown/Knockout Validation:
Design isoform-specific siRNA constructs that target unique regions of each C17orf58 isoform. The commercially available C17orf58 siRNA should be evaluated for isoform specificity before use. Implement these in validation experiments where selective knockdown of specific isoforms can demonstrate antibody specificity. Alternatively, CRISPR-Cas9 genome editing can be employed to create isoform-specific knockout cell lines as definitive controls.

Immunoprecipitation Combined with Mass Spectrometry:
Perform immunoprecipitation using available C17orf58 antibodies followed by high-resolution mass spectrometry. This approach can identify peptide sequences unique to specific isoforms, confirming which isoforms are recognized by a particular antibody. Analysis of post-translational modifications can provide additional distinguishing characteristics between isoforms.

Correlation with Isoform-Specific mRNA Expression:
Design isoform-specific RT-qPCR assays targeting unique exon junctions or sequences. Correlate mRNA expression patterns with protein detection patterns across tissues or experimental conditions. Concordance between isoform-specific mRNA and protein patterns provides supporting evidence for antibody specificity to particular isoforms.

Through this comprehensive approach, researchers can work toward reliable differentiation between C17orf58 isoforms, although the technical challenges should not be underestimated. Documentation of all validation steps and controls is essential for establishing confidence in isoform-specific detection.

What strategies can researchers employ to troubleshoot weak or inconsistent signals when using C17orf58 antibodies?

When encountering weak or inconsistent signals with C17orf58 antibodies, researchers should implement a systematic troubleshooting approach addressing multiple aspects of the experimental workflow:

Protein Expression and Sample Preparation:
First, consider the endogenous expression level of C17orf58 in your experimental system. C17orf58 may be expressed at low levels in certain cell types or tissues, necessitating enrichment strategies. For Western blotting, increase protein loading (up to 30-50 μg) or consider immunoprecipitation to concentrate the target protein. Ensure complete protein extraction by using lysis buffers containing appropriate detergents and protease inhibitors. For comparative studies, validate protein extraction efficiency across different sample types.

Antibody Selection and Optimization:
Re-evaluate the sensitivity and specificity of your chosen C17orf58 antibody. Consider testing multiple antibodies, such as both the mouse monoclonal [OTI6E9] and rabbit polyclonal options . Optimize antibody concentration by performing titration experiments; for Western blotting, test concentrations ranging from 1/500 to 1/5000, deviating from the manufacturer's recommended 1/2000 if necessary . For immunohistochemistry applications, methodically test dilutions between 1:100 to 1:500 .

Detection System Enhancement:
Implement more sensitive detection systems. For Western blotting, switch from standard ECL to enhanced chemiluminescence substrates designed for low-abundance proteins. Consider fluorescent Western blotting for improved quantitative analysis and sensitivity. For immunohistochemistry or immunofluorescence, evaluate signal amplification systems such as tyramide signal amplification (TSA) or polymer-based detection systems that provide multiple enzyme molecules per binding event.

Protocol Optimization Matrix:
Develop a systematic optimization matrix addressing multiple variables:

• For Western blotting: Test different transfer conditions (wet vs. semi-dry), membrane types (PVDF vs. nitrocellulose), blocking reagents (BSA vs. milk), and incubation times (1 hour vs. overnight)

• For IHC/IF: Evaluate fixation methods (paraformaldehyde vs. methanol), antigen retrieval conditions (citrate vs. EDTA buffers, different pH values), blocking solutions (serum vs. protein-based), and mounting media (with vs. without anti-fade)

Positive Control Implementation:
Incorporate reliable positive controls into every experiment. Consider using HEK-293T cells transfected with C17orf58 cDNA as a positive control for Western blotting, as demonstrated in the literature . For tissue staining, identify tissues known to express C17orf58 based on available data from resources like The Human Protein Atlas. These controls serve as essential references for troubleshooting and protocol optimization.

Through methodical application of these troubleshooting strategies, researchers can significantly improve detection of C17orf58 protein, leading to more consistent and reliable experimental outcomes. Careful documentation of all optimization steps will facilitate reproducibility and provide valuable reference for future experiments.

How can researchers integrate C17orf58 antibody-based data with genomic and proteomic datasets for comprehensive protein characterization?

Integrating antibody-based data with genomic and proteomic datasets enables comprehensive characterization of proteins like C17orf58. This multi-omics approach provides richer insights into protein function, regulation, and relevance in biological systems:

Correlation of Expression Across Multiple Platforms:
Begin by establishing a correlation framework between antibody-based detection of C17orf58 protein levels and corresponding mRNA expression data from RNA-seq or microarray studies. Quantify C17orf58 protein expression using validated Western blot or immunohistochemistry protocols , then compare with transcriptomic data from the same samples or cells. Discrepancies between protein and mRNA levels may suggest post-transcriptional regulation mechanisms worthy of further investigation. Extend this correlation analysis to include publicly available datasets from resources like TCGA (The Cancer Genome Atlas) or GTEx (Genotype-Tissue Expression) to place your findings in a broader biological context.

Multi-Modal Protein Characterization:
Implement a complementary set of antibody-dependent and antibody-independent techniques to characterize C17orf58:

• Use antibody-based methods (Western blot, IP, IHC, IF) for detection, localization, and semi-quantitative analysis

• Complement with mass spectrometry-based proteomics for unbiased identification of protein isoforms, post-translational modifications, and interaction partners

• Consider targeted proteomic approaches such as Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM) for absolute quantification

Interaction Network Analysis:
Employ immunoprecipitation with C17orf58 antibodies followed by mass spectrometry (IP-MS) to identify interaction partners. Visualize and analyze the resulting protein-protein interaction networks using tools like STRING, Cytoscape, or NDEx. Cross-reference these experimental interaction data with predicted interactions from bioinformatic analyses. Map interaction partners to cellular pathways to generate hypotheses about C17orf58 function. Consider validation of key interactions using proximity ligation assays or co-immunoprecipitation with antibodies to the partner proteins.

Functional Genomics Integration:
Correlate C17orf58 protein abundance and localization data with functional genomic datasets:

• CRISPR screens to identify genetic dependencies related to C17orf58

• ChIP-seq data to understand transcriptional regulation of C17orf58

• GWAS studies that may implicate C17orf58 in disease susceptibility

Develop a computational framework for integrating these diverse data types, potentially using machine learning approaches to identify patterns and generate predictive models of C17orf58 function .

Spatiotemporal Analysis:
Combine immunohistochemistry or immunofluorescence data showing C17orf58 localization with spatial transcriptomics or imaging mass spectrometry data. This integration provides valuable context about the microenvironment in which C17orf58 functions and may reveal tissue-specific or cell-type-specific roles. Timeline studies across development or disease progression can add temporal dimension to this characterization.

Through methodical integration of antibody-based data with these complementary approaches, researchers can build a comprehensive understanding of C17orf58 biology that transcends the limitations of any single technique or dataset.