C3orf38 Antibody

What is C3orf38 and what are its known biological functions?

C3orf38 (chromosome 3 open reading frame 38) is a 329 amino acid protein that is primarily involved in apoptosis regulation . The protein is encoded by a gene that maps to human chromosome 3p11.1, a region that contains numerous tumor suppressor genes and is frequently deleted in various cancer types .

C3orf38 is localized predominantly in the nucleus and plays a role in the positive regulation of apoptotic processes . While comprehensive expression profiling is still ongoing, C3orf38 has been detected in various human tissues and cell lines, including fetal liver, HT-1376, U87-MG, and Caco-2 cells .

From an evolutionary perspective, the C3orf38 gene is located on chromosome 3, which contains approximately 214 million bases encoding over 1,100 genes, including a chemokine receptor gene cluster and various cancer-related loci . The chromosome 3 region has been noted for its importance in cancer research, as particular regions of its short arm are frequently deleted in many types of cancer cells .

What criteria should researchers consider when selecting a C3orf38 antibody for their specific application?

Selecting the appropriate C3orf38 antibody requires careful consideration of several experimental parameters:

Application compatibility: Different antibodies are optimized for specific applications. For C3orf38 research, consider whether your primary application is Western blot (WB), immunofluorescence (IF), immunohistochemistry (IHC), or ELISA . For example, antibody 25510-1-AP from Proteintech is recommended for WB (1:500-1:1000), IF/ICC (1:20-1:200), and ELISA applications .

Species reactivity: Verify that the antibody recognizes C3orf38 in your species of interest. Available antibodies show different reactivity patterns:

• Human-specific: Boster Bio M17229, Assay Genie CAB20935

• Human and mouse: Proteintech 25510-1-AP

• Human, mouse, and rat: Sigma-Aldrich HPA034737

Antibody type: Choose between monoclonal and polyclonal antibodies based on your research needs:

• Monoclonal antibodies (like Abcam's EPR12512) offer high specificity for a single epitope

• Polyclonal antibodies (like Proteintech's 25510-1-AP) recognize multiple epitopes and may provide stronger signals

Clonality and host species: Consider the host species (typically rabbit) and clonality when designing experiments that involve multiple antibodies to avoid cross-reactivity issues .

Validation data: Examine the manufacturer's validation data, including Western blot images, immunofluorescence patterns, and positive control samples. For example, Abcam's EPR12512 antibody has been validated in HT-1376, U87-MG, Caco-2 cell lysates, and human fetal liver tissue .

How can researchers validate a C3orf38 antibody for their experimental system?

Thorough validation of C3orf38 antibodies is critical for generating reliable research data. The validation process should include:

Positive and negative controls:

• Use cell lines with known C3orf38 expression as positive controls. Based on available data, L02 cells, Neuro-2a cells, K-562, U-937, HEL, HT-1376, U87-MG, and Caco-2 cells have been used successfully .

• Consider using knockout or knockdown models as negative controls to confirm specificity.

Antibody titration:

• Perform a dilution series to determine optimal antibody concentration for your specific application. For Western blot applications, most C3orf38 antibodies work well in the 1:500-1:2000 range .

• For immunofluorescence, starting dilutions of 1:20-1:200 are commonly recommended .

Cross-reactivity assessment:

• Test the antibody in tissues or cells from multiple species if cross-species reactivity is claimed.

• Verify specificity using peptide competition assays or pre-adsorption with the immunizing peptide.

Multiple detection methods:

• Compare results across different techniques (e.g., Western blot, immunofluorescence, and immunohistochemistry).

• For C3orf38, confirm that the detected protein shows the expected molecular weight of approximately 35-38 kDa .

Reproducibility testing:

• Perform at least three independent experiments to ensure consistent results.

• Document batch-to-batch variations if using different lots of the same antibody.

What are the optimal conditions for Western blot detection of C3orf38?

Western blot detection of C3orf38 requires careful optimization of several parameters:

Sample preparation:

• Effective lysis buffers: Standard RIPA buffer with protease inhibitors has been effective for extracting C3orf38 from various cell types.

• Recommended cell types for positive controls: L02 cells, Neuro-2a cells, K-562, U-937, and HEL cells have shown consistent C3orf38 expression .

Electrophoresis and transfer conditions:

• Gel percentage: 10-12% SDS-PAGE gels are appropriate for resolving the 35-38 kDa C3orf38 protein.

• Transfer method: Semi-dry or wet transfer systems can be used with PVDF or nitrocellulose membranes.

Antibody incubation:

• Primary antibody dilutions: The recommended dilution range for C3orf38 antibodies in Western blot applications is 1:500-1:2000 .

• Incubation conditions: Overnight incubation at 4°C typically yields optimal results.

• Secondary antibody selection: Use an anti-rabbit IgG HRP-conjugated secondary antibody for most commercially available C3orf38 antibodies.

Detection system:

• Enhanced chemiluminescence (ECL) systems have been successfully used for detecting C3orf38.

• Expected band size: The observed molecular weight for C3orf38 is 35-37 kDa .

Troubleshooting non-specific bands:

• If additional bands are observed, increase the blocking time or concentration of blocking agent.

• Consider using freshly prepared samples, as C3orf38 may be subject to degradation.

• Optimize antibody concentration through titration experiments.

How can researchers optimize immunofluorescence protocols for C3orf38 detection?

Successful immunofluorescence detection of C3orf38 requires attention to the following methodological details:

Fixation and permeabilization:

• Recommended fixative: 4% paraformaldehyde for 15-20 minutes at room temperature.

• Permeabilization agent: 0.1-0.2% Triton X-100 for 10 minutes is suitable for nuclear protein detection.

Blocking conditions:

• Use 5-10% normal serum (from the same species as the secondary antibody) or BSA.

• Block for at least 1 hour at room temperature to minimize background signal.

Antibody concentration and incubation:

• Primary antibody dilution: The recommended range for IF/ICC is 1:20-1:200 .

• Incubation time: Overnight incubation at 4°C typically yields the best results.

• Secondary antibody selection: Use fluorophore-conjugated secondary antibodies appropriate for your imaging system.

Controls and counterstaining:

• Include a nuclear counterstain such as DAPI to verify the expected nuclear localization of C3orf38 .

• Consider including a cytoskeletal marker as a reference for cellular morphology.

Imaging and analysis:

• C3orf38 should show predominantly nuclear localization .

• For quantitative analysis, consider using standardized imaging parameters and automated analysis software.

Multiplexing considerations:

• When performing multiplex immunofluorescence (MIF), careful selection of primary antibodies from different host species is essential.

• The protocol used in research involving PD-L1/CD3 and CD2/CD3 MIF can be adapted for C3orf38 co-localization studies with relevant markers .

How is C3orf38 implicated in cancer research and what methodologies are used to study this relationship?

C3orf38's potential role in cancer biology stems from its involvement in apoptosis regulation and its location on chromosome 3, a region frequently altered in various cancers .

Genomic alterations:
Chromosome 3, particularly the short arm where C3orf38 is located (3p11.1), contains several tumor suppressor genes and is frequently deleted in many cancer types . Research methodologies to investigate these genomic alterations include:

• Copy number variation (CNV) analysis using OncoScan® arrays to detect deletions or amplifications .

• Molecular inversion probe (MIP) technology for detailed genomic characterization .

• Next-generation sequencing to identify mutations or expression changes in C3orf38.

Expression analysis in cancer tissues:
Studies have examined C3orf38 expression across different cancer types using:

• Immunohistochemistry on tissue microarrays to assess protein expression levels.

• RNA sequencing or microarray analysis to evaluate gene expression changes.

• Western blot analysis of cancer cell lines and patient-derived samples.

Functional studies:
To investigate C3orf38's role in apoptosis regulation in cancer:

• Gene silencing using siRNA or CRISPR-Cas9 technology.

• Overexpression studies to assess effects on cell proliferation and apoptosis.

• Cytotoxicity assays to measure cell death responses, similar to the NAPQI cytotoxicity experiments that identified SNPs in linkage disequilibrium on chromosome 3 .

Clinical correlations:
Researchers have explored associations between C3orf38 expression and:

• Patient survival outcomes

• Treatment responses

• Disease progression markers

One specific area of interest is the potential involvement of C3orf38 in Epstein-Barr virus-associated primary nodal T/NK-cell lymphoma, which shows distinct molecular signatures and copy number changes .

What role does C3orf38 play in immune regulation and how can researchers investigate this function?

C3orf38, also described as an immune regulator, appears to modulate immune responses and maintain immune homeostasis . This function suggests potential involvement in autoimmune diseases, cancer, and other inflammatory conditions . Researchers can investigate this aspect using several approaches:

Expression profiling in immune cells:

• Flow cytometry to quantify C3orf38 expression in different immune cell populations.

• Single-cell RNA sequencing to examine cell-type-specific expression patterns.

• Immune cell activation studies to monitor changes in C3orf38 expression following stimulation.

Protein-protein interaction studies:

• Co-immunoprecipitation assays to identify binding partners of C3orf38 in immune cells.

• Proximity ligation assays to visualize protein interactions in situ.

• Yeast two-hybrid screening to discover novel interactors.

Functional assays:

• Cytokine production measurement following C3orf38 modulation.

• Cell migration and adhesion assays to assess effects on immune cell trafficking.

• T-cell activation and proliferation studies.

Genetic association studies:

• Analysis of SNPs in the C3orf38 gene region and their association with immune-related disorders.

• Genome-wide association studies (GWAS) to identify correlations with autoimmune diseases.

In vivo models:

• C3orf38 knockout or transgenic mouse models to study immune system development and function.

• Disease models to assess the impact of C3orf38 modulation on immune-mediated pathologies.

The multiplexed immunofluorescence techniques used for PD-L1/CD3 and CD2/CD3 analysis, as described in some research, could be adapted to study C3orf38 co-expression with immune markers .

What are common challenges in C3orf38 antibody-based experiments and how can they be addressed?

Researchers working with C3orf38 antibodies may encounter several challenges. Here are systematic approaches to address common issues:

Weak or absent signal in Western blot:

• Increase protein loading (start with 20-30 μg of total protein).

• Optimize antibody concentration - try the more concentrated end of the recommended range (1:500 for most C3orf38 antibodies) .

• Extend primary antibody incubation time to overnight at 4°C.

• Use enhanced chemiluminescence substrates with higher sensitivity.

• Verify sample preparation - ensure effective lysis and minimal protein degradation.

• Check expression levels in your cell type; consider using K-562, U-937, HEL, HT-1376, U87-MG, or Caco-2 cells as positive controls .

Multiple bands or non-specific binding:

• Increase blocking time and concentration (try 5% BSA instead of standard blocking buffer).

• Perform more stringent washing steps (increase number and duration of washes).

• Titrate the antibody to a more dilute concentration.

• Use freshly prepared samples to minimize degradation products.

• Consider the possibility of post-translational modifications or alternative splice variants.

Poor signal in immunofluorescence:

• Optimize fixation and permeabilization conditions for nuclear proteins.

• Increase antibody concentration (start at 1:20 dilution for IF/ICC applications) .

• Extend primary antibody incubation time to 48 hours at 4°C for difficult-to-detect targets.

• Use signal amplification methods (e.g., tyramide signal amplification).

• Try antigen retrieval methods if using fixed tissues.

High background in immunohistochemistry:

• Increase blocking time and concentration.

• Dilute the primary antibody further.

• Include additional washing steps.

• Use more specific secondary antibodies.

• Consider using automated staining platforms for more consistent results.

Inconsistent results between experiments:

• Standardize all protocol steps, including sample preparation, blocking, and washing.

• Document lot numbers of antibodies and reagents.

• Prepare aliquots of antibodies to avoid freeze-thaw cycles.

• Include positive and negative controls in each experiment.

• Maintain consistent imaging parameters across experiments.

How can researchers distinguish between specific and non-specific signals when studying C3orf38?

Distinguishing between specific and non-specific signals is crucial for accurate data interpretation in C3orf38 research:

Validation controls:

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to block specific binding sites.

• Knockout/knockdown controls: Use CRISPR or siRNA to reduce C3orf38 expression and confirm signal specificity.

• Antibody omission: Perform the same protocol without primary antibody to identify secondary antibody non-specific binding.

• Isotype controls: Use non-specific IgG from the same species as the primary antibody to identify Fc receptor binding.

Signal characteristics:

• Molecular weight verification: Authentic C3orf38 should appear at 35-37 kDa in Western blots .

• Subcellular localization: Genuine C3orf38 signal should be predominantly nuclear .

• Signal pattern: Compare with published literature and manufacturer's validation data (e.g., patterns seen in HT-1376, U87-MG, and Caco-2 cells) .

Multiple antibody approach:

• Use antibodies from different sources that recognize distinct epitopes of C3orf38.

• Compare signals from monoclonal and polyclonal antibodies - true signals should appear in both.

• Use antibodies raised against different regions of C3orf38 to confirm target identity.

Correlative techniques:

• Combine antibody-based detection with mRNA expression analysis.

• Use mass spectrometry to confirm protein identity in immunoprecipitated samples.

• Employ proximity ligation assays to verify protein interactions.

Quantitative analysis:

• Compare signal intensity with known expression levels in control samples.

• Use quantitative Western blotting with standard curves.

• Apply digital image analysis to quantify signal-to-noise ratios.

What emerging techniques are being developed for studying C3orf38 expression and function?

Several cutting-edge technologies are enhancing our ability to study C3orf38:

Single-cell technologies:

• Single-cell RNA sequencing to map C3orf38 expression across diverse cell populations.

• Mass cytometry (CyTOF) for high-dimensional protein profiling, including C3orf38 expression in relationship to other cellular markers.

• Spatial transcriptomics to analyze C3orf38 expression in tissue context while preserving spatial information.

Advanced imaging techniques:

• Super-resolution microscopy to visualize C3orf38's precise subcellular localization.

• Multiplexed immunofluorescence with spectral unmixing, as described in literature for other proteins, allowing simultaneous detection of C3orf38 and multiple markers .

• Live-cell imaging with tagged C3orf38 to monitor dynamic processes.

CRISPR-based methods:

• CRISPR activation/inhibition systems for precise modulation of C3orf38 expression.

• CRISPR screens to identify genetic interactions with C3orf38.

• Base editing or prime editing for studying specific mutations in C3orf38.

Protein interaction mapping:

• BioID or APEX proximity labeling to identify proteins in close proximity to C3orf38.

• Hydrogen-deuterium exchange mass spectrometry to map structural dynamics and interaction surfaces.

• Crosslinking mass spectrometry to identify direct binding partners.

Functional genomics approaches:

• Chromatin immunoprecipitation (ChIP) assays similar to those used in related studies to investigate transcription factor binding at C3orf38 regulatory regions .

• ATAC-seq to examine chromatin accessibility at the C3orf38 locus.

• Hi-C and other chromosome conformation capture techniques to study the 3D genomic context of C3orf38.

How can researchers integrate C3orf38 findings into broader molecular pathways and disease mechanisms?

Integrating C3orf38 research into wider biological contexts requires multidisciplinary approaches:

Pathway analysis:

• Use bioinformatics tools to place C3orf38 within known signaling pathways related to apoptosis regulation.

• Perform gene set enrichment analysis (GSEA) on datasets where C3orf38 expression is altered.

• Construct protein-protein interaction networks centered on C3orf38.

Multi-omics integration:

• Combine transcriptomics, proteomics, and metabolomics data to build comprehensive models of C3orf38 function.

• Correlate genomic alterations in the C3orf38 locus with transcriptomic and proteomic changes.

• Apply machine learning approaches to identify patterns in multi-omics datasets including C3orf38.

Disease association studies:

• Analyze C3orf38 expression across patient cohorts with various diseases, particularly those with immune dysregulation or cancer.

• Investigate associations between C3orf38 SNPs and disease susceptibility or treatment responses.

• Examine C3orf38 alterations in the context of chromosome 3 deletions common in certain cancers.

Therapeutic targeting strategies:

• Explore the potential of C3orf38 as a biomarker for disease progression or treatment response.

• Investigate whether modulating C3orf38 function could have therapeutic applications in diseases where apoptosis regulation is important.

• Develop tools to target C3orf38 or its interacting partners as potential therapeutic approaches.

Evolutionary conservation analysis:

• Compare C3orf38 structure and function across species to identify conserved domains essential for its activity.

• Study C3orf38 in model organisms to gain insights into its fundamental biological roles.

• Examine how C3orf38 function has evolved, particularly in relation to immune system development.