C3orf38 Antibody, HRP conjugated

What is C3orf38 protein and what cellular functions does it perform?

C3orf38 (Chromosome 3 open reading frame 38) is a nuclear-localized protein involved in positive regulation of apoptotic processes. The protein functions as an immune regulator, playing key roles in modulating immune responses and maintaining immune homeostasis. Its involvement in these fundamental cellular processes suggests potential significance in autoimmune diseases, cancer biology, and inflammatory conditions . The protein is encoded by a gene 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 . Structurally, C3orf38 has a calculated molecular weight of 37 kDa, though observed weights in experimental settings typically range from 35-38 kDa depending on the cell type and detection method used .

What are the typical applications for C3orf38 antibodies with HRP conjugation?

C3orf38 antibodies with HRP (horseradish peroxidase) conjugation are primarily designed for direct detection applications that eliminate the need for secondary antibodies. The most common validated applications include:

• Western Blot (WB): Recommended dilutions range from 1:300-5000, with most manufacturers suggesting 1:500-1000 for optimal results

• ELISA: Typically used at dilutions of 1:500-1000

• Immunohistochemistry (IHC-P): Applicable for paraffin-embedded sections at 1:200-400 dilutions

• Immunohistochemistry (IHC-F): Suitable for frozen sections at 1:100-500 dilutions

The direct HRP conjugation provides advantages in reducing background signal and cross-reactivity issues that can occur with secondary antibody systems, making these reagents particularly valuable for complex tissue samples or multiplex detection systems .

What species reactivity can be expected from commercially available C3orf38 HRP-conjugated antibodies?

The species reactivity profiles of commercially available C3orf38 HRP-conjugated antibodies vary by manufacturer and clone. Based on validated data:

Most C3orf38 antibodies demonstrate confirmed reactivity with human samples, with some also validating mouse reactivity . When using these antibodies with species other than those explicitly validated, preliminary cross-reactivity testing is strongly recommended to confirm specificity before proceeding with experimental applications.

How should optimal working dilutions be determined for C3orf38 HRP-conjugated antibodies in Western blot applications?

Determining optimal working dilutions for C3orf38 HRP-conjugated antibodies requires a systematic titration approach:

• Begin with a broad dilution series based on manufacturer recommendations (typically 1:300-1:5000 for WB applications)

• Prepare identical membrane strips loaded with the same amount of protein from positive control samples (e.g., L02 cells or Neuro-2a cells for C3orf38)

• Incubate each strip with a different antibody dilution under identical conditions

• Process all strips simultaneously with the same detection system

• Compare signal-to-noise ratios across all dilutions, selecting the concentration that provides maximum specific signal with minimal background

When optimizing, consider that higher antibody concentrations may increase background signal, while excessive dilution can reduce detection sensitivity. For C3orf38, many researchers find that a 1:500 dilution provides an optimal balance for most experimental systems, though this should be validated in your specific experimental context . Always include both positive controls (cells known to express C3orf38) and negative controls (ideally knockout cells) in optimization experiments to ensure specificity .

What are the recommended storage conditions to maintain antibody performance and prevent loss of HRP activity?

To maintain optimal performance of C3orf38 HRP-conjugated antibodies:

• Store at -20°C as recommended by all manufacturers

• Prepare multiple small-volume aliquots upon receiving the antibody to minimize freeze-thaw cycles

• Include cryoprotectants in storage buffer (most commercial preparations include 50% glycerol for this purpose)

• Avoid repeated freeze-thaw cycles which particularly damage HRP enzyme activity

• For working solutions, store at 4°C and use within 1-2 weeks

• Protect from prolonged light exposure during storage and handling

Storage buffer composition is critical for maintaining both antibody binding capacity and HRP enzymatic activity. Most commercial preparations contain stabilizing components such as 0.01M TBS (pH 7.4) with 1% BSA and preservatives like 0.03% Proclin300 or 0.02% sodium azide . Note that sodium azide can inhibit HRP activity if used at high concentrations, but the low levels in storage buffers are generally compatible with short-term storage of HRP-conjugated antibodies .

What controls are essential when validating C3orf38 antibody specificity for research applications?

Rigorous validation of C3orf38 antibodies requires multiple complementary controls:

• Genetic Controls:

  • Wild-type (WT) vs. C3orf38 knockout (KO) cell comparison is the gold standard for specificity validation

  • If KO cells are unavailable, CRISPR-Cas9 or siRNA knockdown samples serve as alternatives

• Expression Controls:

  • Cells with confirmed high C3orf38 expression (e.g., K-562, U-937, HEL for human samples)

  • Cells with confirmed low or undetectable expression

  • Recombinant C3orf38 protein as positive control (especially using the immunogen range 21-120/329)

• Technical Controls:

  • Primary antibody omission to assess secondary antibody non-specific binding

  • Isotype control (rabbit IgG at equivalent concentration) to identify non-specific binding

  • Peptide competition/blocking with immunizing peptide to confirm epitope specificity

• Visualization Controls for Immunostaining:

  • Mosaic plating of positive and negative cells labeled with different cell trackers allows direct side-by-side comparison within the same field of view

  • Co-staining with established cellular markers to confirm expected subcellular localization (nuclear for C3orf38)

When documenting antibody validation, include representative images showing signal in positive samples and absence of signal in negative controls, along with molecular weight confirmation (expected 35-38 kDa for C3orf38) .

How can researchers address inconsistent molecular weight observations when detecting C3orf38?

Inconsistent molecular weight observations for C3orf38 detection can arise from multiple factors:

• Expected Weight Variations:

  • Calculated molecular weight: 37 kDa (329 amino acids)

  • Observed weight ranges from 35-38 kDa across different experimental systems

  • These variations are within normal range and likely reflect post-translational modifications or cell type-specific processing

• Methodological Approaches to Address Inconsistencies:

  • Run gradient gels (4-20%) to improve separation and accurate sizing of the protein

  • Include multiple positive control cell lines (L02, Neuro-2a, K-562, U-937) to establish normal variation range

  • Use recombinant C3orf38 protein as a size standard

  • Apply reducing and non-reducing conditions in parallel to identify potential dimers or complexes

  • Perform phosphatase treatment prior to SDS-PAGE to determine if phosphorylation contributes to observed weight shifts

• Data Interpretation Guidelines:

  • Document all observed molecular weights with clear indication of experimental conditions

  • Consider cell-type specific factors that might influence protein processing

  • Verify questionable bands through additional techniques like mass spectrometry or immunoprecipitation

  • When in doubt, confirm specificity through knockout/knockdown controls

The normal variation in observed molecular weight (35-38 kDa) should not be considered problematic unless bands appear at drastically different sizes or multiple strong bands are observed outside this range .

What are the common sources of non-specific background when using HRP-conjugated C3orf38 antibodies, and how can they be mitigated?

Non-specific background with HRP-conjugated C3orf38 antibodies can arise from several sources:

• Excessive Antibody Concentration:

  • Solution: Perform systematic dilution series to identify optimal antibody concentration

  • For Western blot, start with manufacturer's recommended range (1:500-1:1000)

  • For immunohistochemistry, more dilute solutions may be required (1:200-400)

• Insufficient Blocking:

  • Solution: Optimize blocking conditions using 5% milk or 5% BSA in TBST for Western blots

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

  • Consider alternative blocking agents (casein, commercial blockers) if standard methods fail

• Cross-Reactivity with Related Proteins:

  • Solution: Validate specificity using C3orf38 knockout controls

  • Perform peptide competition assays with the immunizing peptide

  • Consider alternative C3orf38 antibodies targeting different epitopes

• Buffer Compatibility Issues:

  • Solution: Ensure sample buffer does not contain components that interfere with HRP activity

  • Avoid high concentrations of sodium azide, SDS, or reducing agents in working solutions

  • Use manufacturer's recommended buffers (e.g., TBS with 0.1% Tween 20)

• HRP-Specific Background Reduction:

  • Solution: Add 0.05% hydrogen peroxide to blocking buffer to inactivate endogenous peroxidases

  • Use specialized HRP blocking solutions for tissues with high endogenous peroxidase activity

  • Consider shorter substrate incubation times with more sensitive detection systems

Implementation of a systematic optimization approach addressing these factors can significantly enhance signal-to-noise ratio and improve experimental reproducibility with HRP-conjugated C3orf38 antibodies.

How should researchers interpret discrepancies in C3orf38 detection between antibodies from different manufacturers?

When facing discrepancies in C3orf38 detection between antibodies from different manufacturers:

Different antibodies may reveal complementary aspects of C3orf38 biology, particularly if they target distinct domains with different functional roles or accessibility in protein complexes.

How can C3orf38 HRP-conjugated antibodies be effectively used in multiplexed detection systems?

Implementing C3orf38 HRP-conjugated antibodies in multiplexed detection systems requires careful planning:

• Sequential Multiplex Strategy:

  • Apply C3orf38 HRP-conjugated antibody first, develop with substrate

  • Inactivate HRP (using 0.3% hydrogen peroxide or commercial inactivation solutions)

  • Apply subsequent antibodies with different detection systems

  • This approach prevents cross-reactivity but may reduce sensitivity for later antibodies

• Spectral Separation Methods:

  • Pair HRP-conjugated C3orf38 antibody with chromogenic substrates yielding distinct colors

  • Use DAB (brown), AEC (red), or TMB (blue) substrates for contrasting signals

  • Combine with fluorescent-labeled antibodies for other targets after HRP development

  • Document with multi-channel imaging to separate signals

• Technical Considerations:

  • Validate antibody performance individually before attempting multiplexing

  • Optimize blocking between sequential applications to prevent cross-reactivity

  • Consider tyramide signal amplification (TSA) for enhanced sensitivity without increased background

  • Test for potential epitope masking when detecting multiple nuclear proteins like C3orf38

• Data Analysis Approaches:

  • Apply computational unmixing algorithms for overlapping signals

  • Use positive controls for each target in both single and multiplexed conditions

  • Quantify signals separately and analyze co-localization patterns

  • Consider automated image analysis for objective quantification

As C3orf38 exhibits nuclear localization , it can be effectively paired with cytoplasmic or membrane markers in multiplex systems without spatial overlap concerns. This spatial separation facilitates cleaner signal discrimination in complex tissue samples.

What methodological adaptations are necessary when using C3orf38 antibodies for investigating protein-protein interactions?

When investigating C3orf38 protein-protein interactions:

• Immunoprecipitation Optimization:

  • Non-conjugated C3orf38 antibodies are preferable for immunoprecipitation

  • Use gentle lysis buffers to preserve protein complexes (e.g., Pierce IP Lysis Buffer)

  • Optimize antibody-to-bead ratios (typically 1 μg antibody per 30 μL protein A/G beads)

  • Consider cross-linking antibody to beads to prevent antibody co-elution with target proteins

  • Validate IP efficiency using Western blot detection of immunodepleted fractions

• Co-immunoprecipitation Protocol Adaptations:

  • Extend binding incubation times (4°C overnight) to capture transient interactions

  • Include appropriate protease and phosphatase inhibitors to preserve complex integrity

  • Optimize salt and detergent concentrations to maintain specific interactions while reducing background

  • Consider nuclear extraction protocols for effective solubilization of C3orf38 complexes

• Proximity Ligation Assay (PLA) Applications:

  • Combine C3orf38 antibody with antibodies against suspected interacting partners

  • Optimize fixation conditions to preserve nuclear architecture while maintaining epitope accessibility

  • Perform careful controls with known non-interacting nuclear proteins

  • Validate PLA signals using genetic knockout controls to confirm specificity

• Mass Spectrometry Integration:

  • Use C3orf38 antibodies for immunoprecipitation followed by mass spectrometry

  • Compare protein partners identified in different cellular contexts

  • Validate top hits through reciprocal co-immunoprecipitation

  • Analyze interactome in relation to known apoptotic regulation pathways given C3orf38's role in apoptosis

When publishing interaction data, include detailed methodology descriptions and quantify interaction strength through multiple replicates to establish confidence in reported protein-protein interactions.

How can researchers effectively utilize C3orf38 antibodies in studying its role in apoptotic regulation and immune modulation?

To effectively investigate C3orf38's role in apoptotic regulation and immune modulation:

• Experimental Design for Apoptosis Studies:

  • Combine C3orf38 immunodetection with apoptotic markers (cleaved caspases, PARP cleavage, TUNEL)

  • Use time-course experiments following apoptotic stimuli to track C3orf38 expression dynamics

  • Implement C3orf38 overexpression and knockdown approaches to establish causality

  • Apply live-cell imaging with non-HRP conjugated antibodies to monitor C3orf38 localization during apoptosis progression

  • Correlate C3orf38 levels with apoptotic indices across multiple cell types

• Immune Modulation Investigation Approaches:

  • Analyze C3orf38 expression patterns in various immune cell populations

  • Examine expression changes following immune activation or suppression stimuli

  • Correlate C3orf38 levels with production of inflammatory cytokines

  • Investigate effects of C3orf38 modulation on immune cell function and signaling pathways

  • Apply C3orf38 antibodies in flow cytometry to quantify expression levels across immune cell subsets

• Integration with Disease Models:

  • Examine C3orf38 expression in tissue samples from autoimmune diseases

  • Correlate expression patterns with disease severity markers

  • Analyze potential post-translational modifications using phospho-specific antibodies

  • Investigate C3orf38 expression in cancer tissues with varied immune infiltration patterns

  • Apply multiplex immunofluorescence to characterize C3orf38 expression in complex tissue microenvironments

• Mechanistic Investigation Methods:

  • Identify transcription factors regulating C3orf38 expression using ChIP approaches

  • Map protein-protein interactions that modulate C3orf38's apoptotic functions

  • Investigate subcellular redistribution during immune activation or apoptotic signaling

  • Analyze potential isoform-specific functions through targeted antibody applications

The dual roles of C3orf38 in apoptosis regulation and immune modulation suggest it may serve as a molecular link between these processes , making it a valuable target for investigating the coordination of cell death and immune response pathways in various disease contexts.

What emerging applications might benefit from advanced C3orf38 antibody development?

Emerging applications that would benefit from advanced C3orf38 antibody development include:

• Single-Cell Protein Analysis:

  • Development of highly sensitive C3orf38 antibodies compatible with CyTOF mass cytometry

  • Adaptation for single-cell Western blot technologies

  • Integration with microfluidic antibody capture systems

  • These approaches would enable correlation of C3orf38 expression with cellular phenotypes at single-cell resolution

• Live-Cell Imaging Probes:

  • Engineering of non-perturbing antibody fragments (Fabs, nanobodies) against C3orf38

  • Development of photoactivatable or photoconvertible tags for pulse-chase imaging

  • Creation of FRET-based biosensors to monitor C3orf38 conformational changes or interactions

  • These tools would provide dynamic spatiotemporal information about C3orf38 function

• Post-Translational Modification Mapping:

  • Generation of modification-specific antibodies (phospho, ubiquitin, SUMOylation)

  • Development of antibodies recognizing specific C3orf38 conformational states

  • Creation of proximity-dependent labeling tools for identifying transient interaction partners

  • These reagents would reveal regulatory mechanisms controlling C3orf38 activity

• Therapeutic Applications:

  • Engineering of antibodies capable of modulating C3orf38 function

  • Development of antibody-drug conjugates targeting cells with aberrant C3orf38 expression

  • Creation of chimeric antigen receptor constructs for cellular immunotherapy

  • These approaches could translate basic C3orf38 biology into therapeutic strategies

Each of these directions would require significant antibody engineering efforts but could substantially advance understanding of C3orf38's role in normal physiology and disease pathogenesis .

How might improved validation standards for C3orf38 antibodies enhance reproducibility in the field?

Implementation of enhanced validation standards for C3orf38 antibodies would improve research reproducibility through:

• Standardized Knockout Validation:

  • Universal adoption of genetic validation using CRISPR knockout controls

  • Repository development of validated positive and negative control cell lines

  • Standardized reporting format for antibody validation data

  • These measures would establish minimum performance benchmarks for antibody specificity

• Application-Specific Validation:

  • Distinct validation criteria for different applications (WB, IHC, IP, IF)

  • Standardized protocols for each application with defined acceptance criteria

  • Interlaboratory validation networks to confirm antibody performance across environments

  • These approaches would ensure antibodies perform consistently across diverse experimental conditions

• Epitope Mapping Requirements:

  • Comprehensive epitope characterization for all commercial antibodies

  • Assessment of epitope conservation across species for cross-reactivity prediction

  • Evaluation of epitope accessibility in fixed versus live samples

  • This information would enable rational selection of antibodies for specific applications

• Data Repository Development:

  • Centralized database of validation results with raw image data

  • User-contributed performance reviews and optimization protocols

  • Integration with published literature mentioning specific antibody catalog numbers

  • Such resources would accelerate method optimization and highlight reliability issues

Implementation of these enhanced standards would particularly benefit C3orf38 research given its emerging significance in immune regulation and apoptosis pathways , where reliable tools are essential for expanding the current limited understanding of its functions.

What experimental systems would be most informative for investigating the functional significance of C3orf38 in disease contexts?

The most informative experimental systems for investigating C3orf38's functional significance in disease include:

• Advanced Cellular Models:

  • Patient-derived primary cells with disease-relevant mutations or expression patterns

  • Organoid systems modeling tissue-specific C3orf38 functions

  • Co-culture systems examining C3orf38's role in cellular interactions

  • CRISPR-engineered isogenic cell lines with targeted C3orf38 modifications

  • These models would reveal context-dependent functions in physiologically relevant systems

• In Vivo Disease Models:

  • Conditional knockout mouse models allowing tissue-specific C3orf38 deletion

  • Humanized mouse models for immune function studies

  • Patient-derived xenografts for cancer-related investigations

  • These systems would illuminate C3orf38's role in complex disease microenvironments

• High-Throughput Screening Platforms:

  • CRISPR screens identifying genes synthetically lethal with C3orf38

  • Small molecule screens for compounds modulating C3orf38 function or expression

  • Proteomic screens identifying condition-specific interaction partners

  • These approaches would place C3orf38 within broader cellular networks

• Clinical Sample Integration:

  • Tissue microarrays spanning disease progression stages

  • Single-cell analyses of patient samples with varying disease severities

  • Longitudinal sampling during treatment courses

  • These resources would establish clinical relevance of experimental findings

These experimental systems, applied with well-validated C3orf38 antibodies, would advance understanding of its potential roles in autoimmune diseases, cancer, and inflammatory conditions, as suggested by its involvement in immune regulation and apoptotic processes . Particular focus should be placed on diseases affecting chromosome 3, where C3orf38 is located and which contains numerous disease-associated loci .