C3orf38 Antibody, FITC conjugated

What is C3orf38 and why is it important for research?

C3orf38 (Chromosome 3 open reading frame 38) is a protein encoded by the C3orf38 gene in humans. The gene is located on chromosome 3 (3p11.1) on the forward strand, spanning 18,771 bases from chr3:88,149,959-88,168,729 and containing 3 exons. Common aliases include MGC26717, LOC285237, and FLJ54270 .

The C3orf38 protein is 329 amino acids in length with a predicted molecular weight of 37.0 kDa and an isoelectric point of 6.01. It contains a large domain of unknown function, DUF4518, which is part of the protein family pfam15008, thought to be involved in apoptosis regulation . This potential role in cell death pathways makes C3orf38 a target of interest in studies related to apoptosis, cancer, and diseases where programmed cell death pathways are dysregulated.

What are the typical characteristics of C3orf38 antibodies?

C3orf38 antibodies are typically available as polyclonal or monoclonal antibodies with the following characteristics:

• Reactivity: Most C3orf38 antibodies show reactivity with human and mouse samples

• Applications: Commonly validated for Western Blot (WB), Immunofluorescence (IF)/Immunocytochemistry (ICC), and ELISA

• Dilution recommendations: For Western Blot, typical dilutions range from 1:500-1:1000; for IF/ICC, 1:20-1:200

• Molecular weight detection: Observed molecular weight in Western blot analysis is typically 35-37 kDa, corresponding to the predicted size of the C3orf38 protein

• Positive detection: Confirmed in specific cell lines including L02 cells (human) and Neuro-2a cells (mouse)

What are the advantages of using FITC-conjugated antibodies for detection?

FITC (Fluorescein isothiocyanate) conjugated antibodies offer several advantages for protein detection:

• Direct visualization: Allows direct detection without requiring secondary antibodies, simplifying protocols and reducing potential sources of non-specific binding

• Established fluorophore: FITC has excitation/emission maxima wavelengths of approximately 498 nm / 526 nm, compatible with most fluorescence microscopes and flow cytometers

• Multiplex capability: Can be used in combination with other fluorophores with different excitation/emission spectra for simultaneous detection of multiple targets

• Quantitative analysis: Signal intensity can be measured, allowing for quantitative analysis of expression levels

• Preservation: When properly stored in buffers containing glycerol, protein stabilizers, and appropriate pH (typically PBS with 50% glycerol at pH 7.3), FITC-conjugated antibodies maintain their performance over extended periods

What applications is C3orf38 antibody, FITC-conjugated suitable for?

Based on data from similar antibodies, C3orf38 antibody, FITC-conjugated would be suitable for:

• Immunofluorescence microscopy: For visualizing the expression and localization of C3orf38 in fixed cells, with recommended dilutions typically in the range of 1:20-1:200

• Flow cytometry: For analyzing C3orf38 expression in cell populations, particularly for intracellular staining

• Confocal microscopy: For high-resolution imaging of C3orf38 localization within cellular compartments

• Fluorescence-activated Cell Sorting (FACS): For isolating cell populations based on C3orf38 expression levels

The antibody has demonstrated positive detection in specific cell types, including L02 cells for immunofluorescence applications .

How should I optimize protocols for intracellular staining with C3orf38 antibody, FITC-conjugated?

Optimal intracellular staining with C3orf38 antibody, FITC-conjugated requires careful consideration of fixation and permeabilization methods:

• Fixation options:

  • Paraformaldehyde fixation (2-4% PFA for 10-15 minutes at room temperature)

  • Methanol fixation (100% ice-cold methanol for 10 minutes at -20°C)

• Permeabilization methods:

  • For PFA-fixed cells: 0.1-0.5% Triton X-100 for 5-15 minutes

  • Alternative: 0.1% saponin in buffer containing 1-2% BSA

• Staining considerations:

  • Protect from light to prevent photobleaching of FITC

  • Optimize antibody concentration through titration experiments

  • Include appropriate negative controls (isotype control, unstained cells)

  • For multi-color experiments, consider spectral overlap with other fluorophores

• Protocol recommendations:

  • For flow cytometry: Gentler fixation (1-2% PFA) with saponin permeabilization often preserves cellular properties best

  • For microscopy: Stronger fixation (4% PFA) with Triton X-100 permeabilization provides better morphological preservation

When developing new protocols, comparison of multiple fixation/permeabilization combinations is recommended to determine optimal conditions for your specific experimental system.

What controls should I include when using C3orf38 antibody, FITC-conjugated?

A comprehensive set of controls is essential for experiments using C3orf38 antibody, FITC-conjugated:

• Negative controls:

  • Isotype control: FITC-conjugated antibody of the same isotype (typically Rabbit IgG-FITC) to assess non-specific binding

  • Unstained cells: To establish baseline autofluorescence

  • Biological negative: Cell types with minimal C3orf38 expression

  • Genetic negative: C3orf38 knockdown or knockout cells if available

• Positive controls:

  • Known positive cells: Based on literature, L02 cells and Neuro-2a cells show positive detection with C3orf38 antibodies

  • Overexpression system: Cells transfected with C3orf38 expression vector

• Technical controls for flow cytometry:

  • Single-color controls: For compensation in multicolor experiments

  • FMO (Fluorescence Minus One): Includes all fluorophores except FITC to establish gating boundaries

  • Viability dye: To exclude dead cells which may bind antibodies non-specifically

• Validation controls:

  • Alternative detection method: Confirmation with orthogonal techniques (e.g., Western blot using unconjugated C3orf38 antibody)

  • Peptide competition: Pre-incubation of antibody with immunizing peptide to confirm specificity

Implementing these controls will ensure reliable and interpretable results, allowing proper discrimination between specific signal and background.

How does the DUF4518 domain in C3orf38 affect antibody binding and detection?

The DUF4518 domain in C3orf38 has several characteristics that can influence antibody binding and fluorescent signal detection:

• Structural influence:

  • DUF4518 encompasses the majority of the C3orf38 protein

  • Its three-dimensional structure may create conformational epitopes or mask linear epitopes

  • Different fixation methods might alter these conformational epitopes, affecting antibody binding

• Amino acid composition effects:

  • DUF4518 has a high abundance of histidines and a low abundance of serines according to compositional analysis

  • High histidine content may affect pH sensitivity of antibody binding

  • The domain's isoelectric point (6.49) influences charge-based interactions with antibodies

• FITC signal considerations:

  • FITC fluorescence is pH-sensitive, with optimal emission at slightly alkaline pH

  • If the histidine-rich domain creates localized pH environments, this could affect FITC signal intensity

  • The domain's potential interaction with other cellular proteins might cause steric hindrance, affecting signal detection

When designing experiments, consider using antibodies with known epitope locations relative to the DUF4518 domain, and validate results with antibodies targeting different regions of C3orf38 when possible.

What are the considerations for using C3orf38 antibody, FITC-conjugated in apoptosis research?

When using C3orf38 antibody, FITC-conjugated to study apoptosis regulation, several important factors should be considered:

• Biological context:

  • C3orf38 contains a domain (DUF4518) thought to be involved in apoptosis regulation

  • Design experiments that investigate the relationship between C3orf38 and known apoptosis pathways

• Co-staining strategies:

  • Consider co-staining with markers of apoptosis (e.g., Annexin V, cleaved caspases)

  • Choose compatible fluorophores that don't overlap significantly with FITC (excitation: 498 nm / emission: 526 nm)

• Experimental design:

  • Include appropriate apoptosis inducers (e.g., staurosporine, FasL, UV irradiation)

  • Plan time-course experiments to capture dynamic changes in C3orf38 expression during apoptosis

  • Compare results across different cell types to identify cell-specific effects

• Technical considerations:

  • Be aware that apoptotic cells may have increased autofluorescence, which could affect FITC signal interpretation

  • Address potential fixation artifacts; apoptotic cells are more sensitive to fixation procedures

  • Optimize permeabilization to ensure antibody access to intracellular C3orf38 while maintaining cell integrity

Understanding these factors will help design robust experiments to investigate the role of C3orf38 in apoptosis regulation using FITC-conjugated antibodies.

How can I validate the specificity of C3orf38 antibody, FITC-conjugated in my experimental system?

Validating the specificity of C3orf38 antibody, FITC-conjugated is crucial for reliable results:

• Genetic manipulation approaches:

  • siRNA/shRNA knockdown: Reduce C3orf38 expression and confirm a corresponding decrease in antibody signal

  • CRISPR/Cas9 knockout: Generate C3orf38 knockout cells as a negative control

  • Overexpression: Create cells overexpressing C3orf38 and verify increased signal intensity

• Peptide competition assay:

  • Pre-incubate the antibody with immunizing peptide or recombinant C3orf38 protein

  • If the antibody is specific, the signal should be reduced or eliminated due to competition

• Western blot correlation:

  • Perform Western blot on the same samples used for flow cytometry or immunofluorescence

  • Confirm that the antibody detects a protein of the expected molecular weight (35-37 kDa for C3orf38)

  • Compare relative signal intensities across samples to verify consistency

• Multi-antibody validation:

  • Test multiple antibodies targeting different epitopes of C3orf38

  • Consistent results across different antibodies increase confidence in specificity

• Orthogonal validation:

  • Correlate antibody results with mRNA expression (qPCR or RNA-seq)

  • Consider mass spectrometry-based validation for definitive protein identification

By implementing these validation strategies, you can establish confidence in the specificity of your C3orf38 antibody, FITC-conjugated.

I'm experiencing weak signal with C3orf38 antibody, FITC-conjugated. How can I improve this?

If you're experiencing weak signal with C3orf38 antibody, FITC-conjugated, here are systematic approaches to improve detection:

• Antibody concentration optimization:

  • Titrate the antibody to find the optimal concentration

  • Start with the manufacturer's recommended dilution (typically 1:20-1:200 for IF/ICC applications)

  • Test higher concentrations cautiously to avoid increasing non-specific binding

• Improve cell preparation:

  • Ensure high cell viability (>90%) before staining

  • Optimize fixation and permeabilization protocols for intracellular proteins

  • Extend permeabilization time to improve antibody access to intracellular epitopes

• Staining protocol modifications:

  • Increase incubation time (try 45-60 minutes instead of standard 30 minutes)

  • Optimize incubation temperature (4°C, room temperature, or 37°C)

  • Use staining buffer with protein (1-2% BSA) to reduce non-specific binding and improve signal-to-noise ratio

• FITC-specific considerations:

  • Protect samples from light at all steps to prevent photobleaching

  • Use freshly prepared antibody dilutions

  • Optimize pH of staining buffers (FITC fluorescence is optimal at slightly alkaline pH)

• Instrument settings:

  • Optimize voltage settings for the FITC channel

  • Ensure proper instrument maintenance and calibration

  • Consider using a more sensitive detector if available

By systematically addressing these factors, you can optimize your protocol to achieve stronger and more consistent FITC signal when detecting C3orf38.

How should I store and handle C3orf38 antibody, FITC-conjugated to maintain its performance?

Proper storage and handling of C3orf38 antibody, FITC-conjugated is crucial for maintaining antibody performance and fluorophore integrity:

• Long-term storage:

  • Store at -20°C according to manufacturer recommendations

  • Based on similar FITC-conjugated antibodies, typical storage buffer contains PBS with 50% glycerol, stabilizing proteins, and preservatives at pH 7.3

  • Aliquot upon receipt to avoid repeated freeze-thaw cycles

  • Use small aliquots (5-20 μL) based on typical experiment needs

• FITC-specific storage considerations:

  • Protect from light at all times (store in amber tubes or wrap containers in aluminum foil)

  • FITC is sensitive to photobleaching; minimize exposure to light sources

  • FITC fluorescence is optimal at slightly alkaline pH; avoid acidic storage conditions

• Working solution preparation:

  • Thaw aliquots slowly on ice

  • Mix gently by flicking or inverting; avoid vortexing which can damage antibody structure

  • Centrifuge briefly after thawing to collect liquid

  • Prepare dilutions in appropriate buffer just before use

  • Return stock solution to -20°C immediately after use

• Short-term storage:

  • Working dilutions can typically be stored at 4°C for up to 1 week

  • For longer storage of working dilutions, add protein carrier (e.g., 0.5-1% BSA)

  • Always protect from light during all storage conditions

Following these storage and handling guidelines will maximize the performance and shelf-life of your C3orf38 antibody, FITC-conjugated, ensuring consistent results across experiments.

What cell types or tissues show the highest expression of C3orf38 for optimal detection?

Based on available information, the following cell types have demonstrated positive detection with C3orf38 antibodies:

• Confirmed positive cell lines:

  • L02 cells (human liver cell line): Show positive Western blot and immunofluorescence detection

  • Neuro-2a cells (mouse neuroblastoma cell line): Show positive Western blot detection

  • These cell lines would be good positive controls for FITC-conjugated C3orf38 antibody experiments

• Expression considerations:

  • Since C3orf38 may be involved in apoptosis regulation through its DUF4518 domain , expression might be altered during apoptotic processes

  • Treatment with apoptosis inducers might increase C3orf38 expression or alter its subcellular localization

  • Time-course experiments following apoptosis induction could identify optimal detection windows

• Species considerations:

  • C3orf38 antibodies typically show reactivity with human and mouse samples

  • This cross-reactivity suggests conservation of protein structure between these species

  • When working with other species, validation of antibody reactivity is recommended

For initial experiments, L02 cells and Neuro-2a cells would be good starting points for protocol optimization with C3orf38 antibody, FITC-conjugated, based on confirmed detection in previous studies.

How can I quantify results from experiments using C3orf38 antibody, FITC-conjugated?

Quantifying results from experiments using C3orf38 antibody, FITC-conjugated requires application-specific approaches:

• Flow cytometry quantification:

  • Mean/median fluorescence intensity (MFI): Measure the average FITC signal intensity in your population of interest

  • Percent positive cells: Determine the percentage of cells above the threshold set by appropriate controls

  • Staining index: Calculate as (MFI positive - MFI negative) / (2 × SD of MFI negative)

  • Statistical analysis: Compare MFI or percent positive values across different conditions using appropriate statistical tests

• Immunofluorescence microscopy quantification:

  • Integrated density: Measure total fluorescence intensity within defined regions of interest (ROIs)

  • Mean pixel intensity: Assess average signal intensity within cells or subcellular compartments

  • Colocalization analysis: Quantify overlap between C3orf38 and other markers using correlation coefficients

  • Spatial distribution: Analyze nuclear/cytoplasmic ratio or membrane/cytosol distribution

• Normalization approaches:

  • Internal controls: Normalize to housekeeping proteins or invariant cellular structures

  • External standards: Include calibration standards in each experiment

  • Relative quantification: Express results as fold-change relative to control conditions

• Software tools for analysis:

  • Flow cytometry: FlowJo, FCS Express, or Cytobank

  • Microscopy: ImageJ/Fiji, CellProfiler, or manufacturer-specific software

  • Statistical analysis: GraphPad Prism, R, or Python with appropriate packages

By applying these quantification methods, researchers can extract meaningful and reproducible data from experiments using C3orf38 antibody, FITC-conjugated.

What are the best practices for designing multi-color flow cytometry experiments with C3orf38 antibody, FITC-conjugated?

Designing effective multi-color flow cytometry experiments with C3orf38 antibody, FITC-conjugated requires careful planning:

• Panel design considerations:

  • Spectral overlap: FITC (excitation/emission: 498 nm / 526 nm) has potential overlap with other green fluorophores like PE and PerCP

  • Brightness hierarchy: Place FITC on targets with medium-to-high expression levels (like C3orf38) when possible

  • Marker placement: Consider the relative expression levels of other targets when designing panels

• Compensation strategy:

  • Prepare single-color controls for each fluorophore

  • Use compensation beads or cells with high expression of each target

  • Perform compensation before analysis using automatic or manual compensation tools

• Experimental controls:

  • FMO (Fluorescence Minus One) control: Include all fluorophores except FITC

  • Isotype control: FITC-conjugated antibody of the same isotype as the C3orf38 antibody

  • Biological controls: Include known positive and negative samples for C3orf38 expression

• Fixation and permeabilization for intracellular staining:

  • Optimize protocol for simultaneous detection of surface and intracellular markers

  • Consider the effect of fixation on fluorophore brightness and spectral characteristics

  • Test different fixation/permeabilization reagents to maximize signal while maintaining cell integrity

• Data acquisition:

  • Set PMT voltages to position negative populations appropriately

  • Collect sufficient events (typically 10,000-100,000) for robust statistical analysis

  • Consider using acquisition gates to enrich for populations of interest

By following these best practices, researchers can design robust multi-color flow cytometry experiments that provide reliable data on C3orf38 expression in complex cellular systems.

What emerging techniques might enhance detection and analysis of C3orf38?

Several emerging techniques show promise for enhancing C3orf38 detection and analysis:

• Advanced microscopy approaches:

  • Super-resolution microscopy: Techniques like STORM, PALM, or STED could provide nanoscale resolution of C3orf38 localization

  • Light-sheet microscopy: For rapid 3D imaging of C3orf38 distribution in larger specimens with reduced photobleaching

  • Live-cell imaging: Combining FITC-tagged antibody fragments with membrane-permeabilizing peptides for real-time tracking

• High-dimensional cytometry:

  • Mass cytometry (CyTOF): Using metal-tagged antibodies instead of fluorophores to eliminate spectral overlap concerns

  • Spectral flow cytometry: Utilizing full emission spectra rather than bandpass filters to better discriminate fluorophores

  • Imaging flow cytometry: Combining flow cytometry with microscopy to analyze C3orf38 localization patterns at high throughput

• Single-cell analysis:

  • Single-cell RNA-seq with protein detection (CITE-seq): Correlating C3orf38 protein levels with transcriptome-wide expression profiles

  • Single-cell proteomics: Analyzing C3orf38 in the context of the broader proteome at single-cell resolution

  • Spatial transcriptomics: Mapping C3orf38 expression within tissue context with spatial resolution

• Bioinformatic approaches:

  • Machine learning algorithms: For automated identification of C3orf38 expression patterns and correlations with cellular phenotypes

  • Network analysis: Integrating C3orf38 expression data with protein-protein interaction networks to infer functional relationships

  • Multi-omics integration: Combining C3orf38 protein data with genomics, transcriptomics, and metabolomics for comprehensive understanding

These emerging techniques promise to provide deeper insights into C3orf38 biology beyond what is currently possible with standard antibody-based detection methods.

What are the most recent advances in understanding C3orf38 function and regulation?

While the search results provide limited information on very recent advances specifically related to C3orf38 function, we can highlight what is known and potential directions:

• Structural insights:

  • C3orf38 contains a DUF4518 domain that is part of the protein family pfam15008

  • This domain is thought to be involved in apoptosis regulation

  • Further structural studies may reveal how this domain functions at the molecular level

• Potential regulatory mechanisms:

  • The gene contains several identified promoter regions, suggesting complex transcriptional regulation

  • Understanding the factors that bind these promoters could provide insights into when and where C3orf38 is expressed

• Functional implications:

  • The potential role in apoptosis regulation makes C3orf38 a target of interest in cancer research and development biology

  • Its expression in neural cell types (e.g., Neuro-2a cells) might suggest a role in neural development or function

• Research tools development:

  • New antibodies and detection methods continue to be developed, improving our ability to study C3orf38

  • CRISPR/Cas9 gene editing allows for more precise functional studies through knockout and knockin approaches

Future research directions will likely focus on elucidating the specific molecular mechanisms by which C3orf38 contributes to apoptosis regulation and identifying interaction partners that mediate its biological functions.