Recombinant Human UPF0461 protein C5orf24 (C5orf24)

What is C5orf24 and where is it localized in human cells?

C5orf24 (chromosome 5 open reading frame 24) is a protein encoded by the C5orf24 gene located at chromosome locus 5q31.1 in humans. The protein is primarily localized to the nucleus and is highly conserved with orthologs present across various vertebrate species including mammals, birds, reptiles, amphibians, and fish . This evolutionary conservation suggests functional importance. The protein belongs to the UPF0461 family, and its nuclear localization indicates potential roles in gene expression regulation, DNA repair, or other nuclear processes.

What are the structural characteristics of the C5orf24 gene and protein?

The C5orf24 gene spans 26,133 base pairs (chr5:134,833,603-134,859,735) and is composed of two exons and one intron, oriented on the plus strand . Alternative names for this gene include FLJ37562 and LOC134553. The genomic context shows neighboring genes such as DDX46, RPL34P13, and TXNDC15 .

The C5orf24 protein (isoform 1) consists of 188 amino acids with two notable disordered regions at positions 1-20 and 79-142 . The second disordered region contains a series of internal repeats that may be functionally significant. The protein has a predicted molecular weight of approximately 20.1 kDa and an isoelectric point of approximately 10, suggesting it carries a net positive charge at physiological pH .

What transcript variants of C5orf24 have been identified?

Three mRNA transcript variants of the human C5orf24 gene have been characterized:

Transcript variants 1 and 2 both encode the same protein (isoform 1, 188 amino acids), with variant 1 having a longer 5' UTR region. Transcript variant 3 lacks an internal segment, resulting in an alternative translational stop codon and producing a shorter isoform of 155 amino acids (isoform 2) .

What antibodies are available for C5orf24 detection and characterization?

Commercial antibodies for C5orf24 include rabbit polyclonal antibodies such as ab235404, which has been validated for Western blotting (WB) and immunohistochemistry on paraffin-embedded tissues (IHC-P) in both human and mouse samples . The immunogen used corresponds to a recombinant fragment of human UPF0461 protein C5orf24 from amino acid 1 to the C-terminus . When selecting antibodies for C5orf24 research, consider the specific application needs, species reactivity, and whether the antibody recognizes specific isoforms or all variants of the protein.

What is the optimal methodology for detecting C5orf24 protein expression in tissue samples?

For detecting C5orf24 protein expression in tissue samples, a multi-method approach is recommended:

• Immunohistochemistry (IHC-P): For paraffin-embedded tissues, antibodies such as ab235404 have been validated at 1/100 dilution . This method allows visualization of the protein's spatial distribution and cellular localization within tissue architecture.

• Western Blotting: For quantitative assessment, Western blotting with antibodies like ab235404 (1/1000 dilution) can detect C5orf24 in various cell types . The expected band size is approximately 20 kDa, consistent with the predicted molecular weight of the protein .

• Validation Controls: Include appropriate positive controls such as kidney tissue for IHC-P and cell lines like K562, THP-1, U-87 MG, HepG2, or HeLa for Western blotting, as these have demonstrated detectable C5orf24 expression .

• Isoform Specificity: Consider using primers or antibodies that can distinguish between the 188 aa (isoform 1) and 155 aa (isoform 2) variants if isoform-specific expression is being investigated .

How can researchers effectively design experiments to study C5orf24 function?

To effectively study C5orf24 function, consider the following experimental design strategies:

• Gene Silencing/Knockout: Implement CRISPR-Cas9 or RNAi approaches targeting C5orf24. Design guide RNAs or siRNAs specific to conserved regions of the transcript to ensure effective knockdown/knockout. Consider targeting specific exons to affect all or selected transcript variants.

• Overexpression Studies: Create expression vectors containing the C5orf24 coding sequence (with or without tags) for transfection into relevant cell lines. Compare functional outcomes between cells expressing isoform 1 (188 aa) versus isoform 2 (155 aa) .

• Protein Interaction Studies: Employ immunoprecipitation followed by mass spectrometry to identify protein binding partners. Given C5orf24's nuclear localization, focus on nuclear extracts and consider chromatin immunoprecipitation if DNA-binding functions are suspected.

• Transcriptional Regulation: Analyze the promoter region for binding sites of transcription factors like NRF1, E2F, ZF5, and AHR that are predicted to regulate C5orf24 expression . Use reporter assays to confirm these regulatory relationships.

• Functional Assessment: Design phenotypic assays based on cellular processes relevant to nuclear proteins, such as cell cycle progression, DNA damage response, or gene expression regulation.

What is known about C5orf24's potential role in disease pathology, particularly in cancer?

While the exact function of C5orf24 in disease pathology remains under investigation, several lines of evidence suggest potential involvement in cancer:

• Expression in Cancer Cell Lines: Western blot analysis has demonstrated C5orf24 expression in multiple cancer cell lines, including K562 (chronic myelogenous leukemia), THP-1 (monocytic leukemia), U-87 MG (glioblastoma), HepG2 (hepatocellular carcinoma), and HeLa (cervical adenocarcinoma) .

• Correlation Studies: Correlation analysis in colorectal cancer research has identified C5orf24 among genes potentially relevant to cancer pathogenesis, with a correlation P-value of 1.694E-01 and R² of 7.478E-03 . While this correlation is not particularly strong, it suggests possible involvement in cancer-related processes.

• Research Approaches: To investigate C5orf24's role in cancer:

  • Compare expression levels between matched tumor and normal tissues

  • Analyze correlations between expression levels and clinical outcomes

  • Investigate effects of C5orf24 knockdown or overexpression on cancer cell proliferation, migration, and invasion

  • Explore potential interactions with known cancer-related signaling pathways

Current evidence is preliminary, and more targeted studies are needed to establish definitive roles in cancer or other diseases.

How can synchronous gene expression analysis techniques be applied to study C5orf24's functional networks?

Advanced synchronous gene expression analysis techniques can reveal important insights into C5orf24's functional networks:

• Maestro Algorithm Application: The Maestro algorithm offers advantages for studying genes like C5orf24 by detecting orchestrated genetic activities that might be missed by conventional co-expression analysis . This approach:

  • Creates separate nodes for high and low expression patterns of each gene

  • Eliminates errors from dual-expressed nodes that might mask important relationships

  • Identifies co-expression modules from entire sample sets before testing trait associations

• Implementation Strategy:

  • Generate expression data for C5orf24 alongside potential functional partners

  • Apply the Maestro algorithm to identify direction-specific co-expression patterns

  • Validate findings through permutation tests to ensure robustness

  • Split datasets into discovery and validation sets to ensure replicability

• Data Interpretation: When analyzing results, focus on genes that may not show significant differential expression individually but contribute to modules involving C5orf24. This approach may uncover synergistic relationships that traditional differential expression analysis would miss .

What experimental approaches can resolve contradictory findings about C5orf24 function or expression?

When faced with contradictory findings regarding C5orf24 function or expression, the following methodological approaches can help resolve discrepancies:

• Standardized Tissue/Cell Processing: Implement consistent protocols for sample preparation, as variations in sample handling can significantly affect gene expression results . This includes standardized tissue collection, processing times, and preservation methods.

• Technical Replication: Perform experiments using multiple technical platforms (e.g., microarray, RNA-seq, qPCR) to cross-validate expression findings.

• Biological Replication: Increase sample sizes and ensure appropriate statistical power. Split large datasets into discovery and validation cohorts to test replicability of findings .

• Isoform-Specific Analysis: Differentiate between the 188 aa and 155 aa isoforms of C5orf24 , as contradictory results may stem from isoform-specific functions or expression patterns not distinguished in previous studies.

• Context-Dependent Function Assessment: Evaluate C5orf24 function across different cell types, tissue contexts, and experimental conditions, as proteins often exhibit context-dependent roles.

• Computational Meta-Analysis: Apply computational approaches that integrate multiple datasets while accounting for batch effects and methodological differences between studies .

What are the optimal conditions for producing recombinant C5orf24 protein for functional studies?

For producing recombinant C5orf24 protein:

How can researchers effectively analyze C5orf24 expression data across different experimental platforms?

To effectively analyze C5orf24 expression data across different experimental platforms:

• Platform-Specific Normalization:

  • For microarray data: Apply RMA or GCRMA normalization

  • For RNA-seq: Use TPM or RPKM/FPKM normalization

  • For qPCR: Normalize to multiple validated reference genes

• Cross-Platform Integration:

  • Convert all expression values to Z-scores or percentile ranks to facilitate comparisons

  • Implement batch correction methods (ComBat, SVA) to minimize technical variation

  • Use rank-based methods for meta-analysis to reduce platform-specific biases

• Validation Strategies:

  • Confirm key findings using orthogonal methods (e.g., RNA-seq findings with qPCR)

  • Include identical reference samples across experiments as internal controls

  • When comparing to published data, account for potential batch effects

• Statistical Considerations:

  • Apply FDR correction for multiple testing

  • Use non-parametric tests when distributions cannot be assumed to be normal

  • Consider mixed-effects models to account for both biological and technical variability

• Isoform-Specific Analysis:

  • Design primers or probes that distinguish between transcript variants 1, 2, and 3

  • For RNA-seq, use algorithms specifically designed for isoform quantification (e.g., Salmon, RSEM)

What considerations should be made when interpreting C5orf24 antibody-based experimental results?

When interpreting results from C5orf24 antibody-based experiments, consider the following factors:

• Antibody Specificity:

  • Validate specificity using positive controls (e.g., recombinant C5orf24) and negative controls (e.g., C5orf24 knockout cells)

  • Determine if the antibody recognizes both isoform 1 (188 aa) and isoform 2 (155 aa)

  • Check for potential cross-reactivity with similar proteins through immunoprecipitation followed by mass spectrometry

• Application-Specific Considerations:

  • For Western blotting: Verify the observed molecular weight matches the predicted 20 kDa

  • For IHC-P: Assess staining patterns against known localization (primarily nuclear)

  • For immunoprecipitation: Confirm enrichment of the target protein by mass spectrometry

• Experimental Controls:

  • Include appropriate positive controls (e.g., kidney tissue for IHC-P, K562 or HeLa cells for Western blot)

  • Use blocking peptides to confirm specificity of staining

  • Include technical replicates to assess consistency

• Quantification Methods:

  • For Western blot: Use densitometry with appropriate normalization (total protein or housekeeping proteins)

  • For IHC: Apply standardized scoring systems (H-score, Allred score) and blinded assessment by multiple observers

  • Document image acquisition settings and processing steps for reproducibility

What are the most effective strategies for studying the transcriptional regulation of C5orf24?

To effectively study the transcriptional regulation of C5orf24:

• Promoter Analysis:

  • Perform in silico analysis of the promoter region (GXP_7545710) to identify binding sites for transcription factors like NRF1, E2F, ZF5, and AHR

  • Create reporter constructs containing the C5orf24 promoter driving luciferase or GFP expression

  • Generate truncated or mutated promoter variants to pinpoint key regulatory elements

• Transcription Factor Binding Studies:

  • Conduct chromatin immunoprecipitation (ChIP) assays for predicted transcription factors

  • Perform electrophoretic mobility shift assays (EMSA) to confirm direct binding

  • Use transcription factor overexpression or knockdown to assess effects on C5orf24 expression

• Epigenetic Regulation:

  • Analyze DNA methylation patterns in the promoter region using bisulfite sequencing

  • Perform ChIP for histone modifications associated with active (H3K4me3, H3K27ac) or repressed (H3K27me3, H3K9me3) chromatin

  • Test effects of HDAC inhibitors or DNA methyltransferase inhibitors on C5orf24 expression

• Context-Dependent Regulation:

  • Compare regulatory mechanisms across different cell types and tissues

  • Investigate changes in regulation under various physiological or stress conditions

  • Examine potential enhancer elements that may regulate C5orf24 from distal genomic regions

What emerging technologies might advance our understanding of C5orf24 function?

Emerging technologies that could significantly advance C5orf24 research include:

• Single-Cell Omics:

  • Single-cell RNA-seq to reveal cell type-specific expression patterns of C5orf24

  • Single-cell ATAC-seq to identify cell-specific regulatory elements controlling C5orf24 expression

  • Spatial transcriptomics to map C5orf24 expression within complex tissue architectures

• CRISPR Technologies:

  • CRISPR activation/inhibition (CRISPRa/CRISPRi) for precise modulation of C5orf24 expression

  • CRISPR base editing for introducing specific mutations to study structure-function relationships

  • CRISPR screens to identify genes functionally interacting with C5orf24

• Protein Analysis Technologies:

  • Proximity labeling methods (BioID, APEX) to identify proteins in close proximity to C5orf24 in living cells

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to analyze conformational dynamics

  • Cryo-electron microscopy for structural determination, particularly of protein complexes involving C5orf24

• Systems Biology Approaches:

  • Integration of multi-omics data using AI/machine learning to predict C5orf24 functions

  • Network medicine approaches to position C5orf24 within disease-relevant pathways

  • Synchronous gene expression analysis using algorithms like Maestro to identify functional modules

How might researchers design studies to elucidate the potential roles of C5orf24 in disease pathogenesis?

To investigate C5orf24's role in disease pathogenesis:

• Comprehensive Expression Profiling:

  • Analyze C5orf24 expression across tissue and cancer databases (TCGA, GTEx)

  • Compare expression between matched disease and normal tissues with attention to both isoforms

  • Correlate expression levels with clinical parameters and survival outcomes

• Functional Genomics Approaches:

  • Conduct CRISPR knockout/knockdown followed by RNA-seq to identify downstream effects

  • Perform rescue experiments with different C5orf24 isoforms to determine functional specificity

  • Use inducible systems to study temporal aspects of C5orf24 function

• Disease Model Systems:

  • Generate C5orf24 transgenic or knockout animal models to study systemic effects

  • Utilize patient-derived organoids or xenografts to study effects in more relevant disease models

  • Develop cell line models with endogenous tagging of C5orf24 to track the protein under different conditions

• Mechanism Identification:

  • Investigate protein-protein interactions specific to disease contexts

  • Examine post-translational modifications that might regulate C5orf24 function

  • Study subcellular localization changes in response to disease-relevant stimuli

• Therapeutic Exploration:

  • Assess C5orf24 as a potential biomarker for disease diagnosis or progression

  • Evaluate the protein as a possible therapeutic target if disease associations are established

  • Develop tools (antibodies, small molecules) to modulate C5orf24 function in disease contexts