C3orf38 Antibody, Biotin conjugated

What is C3orf38 and what is its biological function?

C3orf38 (Chromosome 3 Open Reading Frame 38) is a protein encoded by the C3orf38 gene (Gene ID: 285237) with a predicted molecular weight of approximately 37-38 kDa. This protein is primarily localized in the nucleus and is believed to play a significant role in apoptosis regulation . The protein has 329 amino acids and is identified by the Swiss-Prot accession number Q5JPI3 . Current research indicates that while its precise mechanism remains under investigation, C3orf38 appears to function in cellular death pathways, making it a protein of interest in cancer research and cellular biology studies.

What are the general characteristics of biotin-conjugated C3orf38 antibodies?

Biotin-conjugated C3orf38 antibodies are immunoglobulins that specifically recognize the C3orf38 protein and have been chemically linked to biotin molecules. These antibodies typically:

The biotin conjugation enables secondary detection through streptavidin-based systems, providing enhanced sensitivity and flexibility in detection methods compared to unconjugated antibodies .

What are the major applications for biotin-conjugated C3orf38 antibodies?

Biotin-conjugated C3orf38 antibodies are versatile research tools with several applications:

• Western Blot (WB): Used at dilutions typically ranging from 1:300-1:5000 to detect C3orf38 protein in cell and tissue lysates, with expected bands at approximately 35-37 kDa .

• ELISA: Applied at dilutions of approximately 1:5000-1:10000 for quantitative detection of C3orf38 protein in solution .

• Immunohistochemistry (IHC): Some biotin-conjugated antibodies are suitable for tissue section analysis, though specific protocols may vary by manufacturer .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): While less commonly mentioned for the biotin-conjugated versions specifically, these applications can be performed using appropriate streptavidin-fluorophore conjugates for detection .

The biotin-streptavidin system provides signal amplification, making these conjugated antibodies particularly useful for detecting low-abundance targets like C3orf38 in complex biological samples .

How do different epitope targets of C3orf38 antibodies affect experimental outcomes?

The epitope specificity of C3orf38 antibodies significantly impacts experimental results through several mechanisms:

Different commercial C3orf38 antibodies target various regions of the protein:

• Antibodies targeting amino acids 80-329 region have shown strong reactivity in ELISA applications

• Antibodies directed against the 21-120 amino acid region demonstrate utility in Western blot, ELISA, and immunohistochemistry applications

• Full-length protein targeting (AA 1-327) antibodies may provide different recognition patterns

Epitope accessibility varies by application. In Western blot, denatured proteins expose most epitopes, while in applications using native proteins (like IF/ICC), only surface-exposed epitopes are accessible. When selecting a biotin-conjugated C3orf38 antibody, researchers should consider whether the target epitope remains accessible after biotin conjugation, as the attachment of biotin molecules may occasionally interfere with antigen recognition depending on conjugation chemistry and epitope proximity .

Additionally, some epitopes may be conserved across species while others are species-specific, explaining the variable cross-reactivity patterns observed among different C3orf38 antibodies .

What factors influence the selection between polyclonal and monoclonal biotin-conjugated C3orf38 antibodies?

The choice between polyclonal and monoclonal biotin-conjugated C3orf38 antibodies should be based on experimental requirements:

How does sample preparation affect the performance of biotin-conjugated C3orf38 antibodies?

Sample preparation significantly influences the performance of biotin-conjugated C3orf38 antibodies across different applications:

For Western Blot applications:

• Cell lysis buffer composition affects protein extraction efficiency and preservation of epitopes

• Complete denaturation of samples enhances epitope accessibility for C3orf38 detection

• Based on available data, cell lines such as HT-1376, U87-MG, Caco-2, L02, and Neuro-2a have been successfully used as positive controls

For Immunohistochemistry and Immunofluorescence:

• Fixation method impacts epitope preservation, with paraformaldehyde fixation generally yielding better results than formalin or methanol fixation (as observed with similar biotin-conjugated antibodies)

• Antigen retrieval methods may be necessary to unmask epitopes in fixed tissues

• Blocking endogenous biotin is critical when using biotin-conjugated antibodies to prevent background signal, especially in biotin-rich tissues

For ELISA applications:

• Coating buffers and blocking solutions must be optimized to prevent non-specific binding

• The recommended dilution ranges (1:5000-1:10000) should be experimentally validated for each specific sample type

Pre-absorption of antibodies with recombinant C3orf38 protein prior to use can be employed as a specificity control in critical applications .

How can researchers optimize signal-to-noise ratio when using biotin-conjugated C3orf38 antibodies in multi-color immunofluorescence studies?

Optimizing signal-to-noise ratio in multi-color immunofluorescence studies using biotin-conjugated C3orf38 antibodies requires several targeted approaches:

• Endogenous biotin blocking: Tissues and cells often contain endogenous biotin that can produce background signals. Implement a sequential blocking protocol:

  • Initial blocking with unconjugated avidin (10-20 μg/mL, 15 minutes)

  • Followed by biotin solution (50 μg/mL, 15 minutes)

  • Standard protein blocking (5% BSA or serum)

• Sequential detection strategy: When using multiple biotin-conjugated antibodies:

  • Apply the first biotin-conjugated C3orf38 antibody

  • Detect with a specific streptavidin-fluorophore

  • Block remaining biotin sites with excess unconjugated streptavidin

  • Apply subsequent biotin-conjugated antibodies with different detection fluorophores

• Spectral optimization: Select detection fluorophores with minimal spectral overlap to reduce bleed-through. For C3orf38 biotin-conjugated antibodies, streptavidin conjugates with the following fluorophores work well:

  • Alexa Fluor 488 for green channel detection

  • Alexa Fluor 647 for far-red detection to avoid autofluorescence

• Titration optimization: Based on IF/ICC dilution recommendations for C3orf38 antibodies (1:20-1:200), perform systematic titration to determine optimal concentration that maximizes specific signal while minimizing background .

• Validation controls: Always include:

  • Secondary-only controls (streptavidin-fluorophore without primary antibody)

  • Samples known to be negative for C3orf38 expression

  • Competitive blocking with recombinant C3orf38 protein

These approaches help overcome the inherent challenges of using biotin-based detection systems in complex immunofluorescence experimental designs .

What are the considerations for using biotin-conjugated C3orf38 antibodies in studying apoptosis mechanisms?

When investigating apoptosis mechanisms using biotin-conjugated C3orf38 antibodies, researchers should consider several specialized factors:

• Timing of fixation: Since C3orf38 is implicated in apoptosis regulation , the protein's expression, localization, and modification state may change rapidly during cell death. Time-course experiments with precise fixation timepoints are crucial.

• Co-localization studies: To understand C3orf38's role in apoptotic pathways:

  • Pair biotin-conjugated C3orf38 antibodies with antibodies against established apoptotic markers (cleaved caspase-3, PARP)

  • Use streptavidin-conjugated far-red fluorophores to avoid spectral overlap with common apoptotic markers

  • Employ confocal microscopy for accurate subcellular localization

• Cell-specific expression patterns: Differential C3orf38 expression has been observed across cell types:

  • Human cell lines like HT-1376, U87-MG, and Caco-2 show detectable expression levels

  • L02 and Neuro-2a cells have been validated for C3orf38 expression studies

• Stimulus-dependent regulation: Design experiments to track C3orf38 dynamics following apoptotic stimuli:

  • Compare intrinsic vs. extrinsic apoptotic pathway induction

  • Monitor nuclear vs. cytoplasmic distribution of C3orf38 during apoptosis progression

  • Consider post-translational modifications that may affect antibody recognition

• Technical considerations for apoptotic cells:

  • Apoptotic cells may exhibit increased autofluorescence

  • Membrane permeability changes during apoptosis may alter antibody accessibility

  • Protein degradation during late apoptosis may reduce C3orf38 detection

These specialized considerations help ensure meaningful results when studying this relatively uncharacterized protein in apoptotic contexts .

How can researchers validate specificity of biotin-conjugated C3orf38 antibodies in their experimental systems?

Comprehensive validation of biotin-conjugated C3orf38 antibodies requires a multi-faceted approach:

• Genetic manipulation strategies:

  • CRISPR/Cas9 knockout of C3orf38: Compare staining patterns between wild-type and knockout samples

  • siRNA-mediated knockdown: Observe reduction in signal proportional to knockdown efficiency

  • Overexpression studies: Confirm increased signal intensity in cells transfected with C3orf38 expression constructs

• Peptide competition assays:

  • Pre-incubate antibody with excess C3orf38 synthetic peptide (the immunogen)

  • Parallel samples tested with competing peptide should show significantly reduced signal

  • Use unrelated peptides as negative controls

• Multi-antibody validation:

  • Test multiple antibodies targeting different C3orf38 epitopes (21-120, 80-329 regions)

  • Compare results between monoclonal (like EPR12512) and polyclonal antibodies

  • Concordant results across different antibodies increase confidence in specificity

• Cross-application validation:

  • If a signal is detected by Western blot at the expected 35-37 kDa size

  • The same samples should show specific staining in immunohistochemistry or immunofluorescence

  • Discrepancies between applications warrant further investigation

• Mass spectrometry confirmation:

  • Immunoprecipitate C3orf38 using the biotin-conjugated antibody

  • Confirm protein identity by mass spectrometry

  • This approach provides definitive validation of antibody specificity

A systematic validation approach ensures that experimental findings truly reflect C3orf38 biology rather than artifacts from non-specific antibody binding .

What are the optimal storage conditions and handling practices for biotin-conjugated C3orf38 antibodies?

Proper storage and handling of biotin-conjugated C3orf38 antibodies is essential for maintaining their performance over time:

Additional handling considerations specific to biotin-conjugated antibodies:

• Avoid using BSA as a carrier protein in solutions if downstream streptavidin detection is planned, as commercial BSA may contain trace biotin

• Monitor signs of degradation such as precipitation, cloudiness, or reduced performance in positive control samples

• Store antibody vials upright to prevent concentration of antibody in the cap during freezing

These storage and handling practices help ensure consistent performance throughout the shelf-life of biotin-conjugated C3orf38 antibodies .

How do researchers troubleshoot weak or absent signals when using biotin-conjugated C3orf38 antibodies?

When encountering weak or absent signals with biotin-conjugated C3orf38 antibodies, a systematic troubleshooting approach should be implemented:

• Sample-related issues:

  • Confirm C3orf38 expression in your sample type (reference positive controls: L02 cells, Neuro-2a cells, HT-1376, U87-MG, Caco-2)

  • Ensure adequate protein loading (25-50 μg total protein typically required for Western blot)

  • Check for protein degradation by examining other proteins in the same sample

  • Verify sample preparation maintains C3orf38 epitope integrity

• Antibody-related factors:

  • Test different antibody concentrations (dilutions from 1:300-1:5000 for WB, 1:20-1:200 for IF/ICC)

  • Consider using a more sensitive detection system (enhanced chemiluminescence for WB)

  • Evaluate antibody quality with positive control samples

  • Determine if biotin conjugation might be interfering with epitope recognition

• Detection system optimization:

  • Ensure streptavidin reagent is functional (test with biotinylated control proteins)

  • Try different streptavidin conjugates (HRP, fluorophores) for optimal detection

  • Increase incubation time with streptavidin conjugate

  • Consider signal amplification systems (tyramide signal amplification)

• Protocol modifications:

  • Extend primary antibody incubation (overnight at 4°C)

  • Optimize antigen retrieval methods for fixed tissues

  • Reduce washing stringency

  • Try different blocking agents to reduce background while preserving specific signal

• Technical controls:

  • Include a housekeeping protein control (β-actin biotin-conjugated antibody)

  • Compare results with non-conjugated C3orf38 antibodies

  • Test freshly prepared buffers and reagents

This methodical approach helps identify and address the specific factors limiting antibody performance in your experimental system .

What are the considerations for quantitative analysis of C3orf38 expression using biotin-conjugated antibodies?

Quantitative analysis of C3orf38 expression using biotin-conjugated antibodies requires rigorous methodological considerations:

• Standardization for Western blot quantification:

  • Use recombinant C3orf38 protein standards at known concentrations

  • Normalize C3orf38 signal to validated loading controls (β-actin)

  • Ensure signal falls within the linear dynamic range of detection system

  • Perform technical replicates (minimum 3) for statistical validity

  • Apply appropriate normalization to account for total protein loading variations

• ELISA-based quantification approaches:

  • Develop a standard curve using recombinant C3orf38 protein

  • Optimal working dilution for biotin-conjugated C3orf38 antibodies in ELISA is typically 1:5000-1:10000

  • Determine limits of detection and quantification for your specific system

  • Address matrix effects by preparing standards in the same buffer as samples

• Flow cytometry considerations:

  • When using biotin-conjugated C3orf38 antibodies for flow cytometry:

    • Titrate antibody concentration to optimize signal-to-noise ratio

    • Use fluorescence minus one (FMO) controls

    • Account for cell autofluorescence and non-specific binding

    • Consider fixation and permeabilization effects on epitope accessibility

• Image-based quantification factors:

  • For immunofluorescence/immunohistochemistry:

    • Standardize image acquisition parameters across all samples

    • Perform automated unbiased quantification using software tools

    • Account for subcellular localization (nuclear vs. cytoplasmic)

    • Use appropriate segmentation methods to distinguish specific signal

• Statistical analysis requirements:

  • Apply appropriate statistical tests based on data distribution

  • Account for biological variability with sufficient biological replicates

  • Consider power analysis to determine required sample size

  • Report both statistical and biological significance