C5orf22 Antibody

What is C5orf22 and why is it studied?

C5orf22 (Chromosome 5 Open Reading Frame 22) is a human protein encoded by a gene located on chromosome 5. While its precise function remains under investigation, it represents one of many uncharacterized proteins (UPFs) that require further study to elucidate their biological roles. Researchers investigate C5orf22 using specific antibodies to understand its expression patterns, subcellular localization, and potential functions in normal and disease states .

What types of C5orf22 antibodies are available for research?

Multiple C5orf22 antibody types exist for research applications:

Most commonly used are rabbit polyclonal antibodies targeting various epitopes of the human C5orf22 protein, with unconjugated versions being predominant for western blotting applications .

How are C5orf22 antibodies generated?

C5orf22 antibodies are typically produced using one of two approaches:

• Synthetic peptide immunization: Antibodies like ABIN6750628 are generated using synthetic peptides corresponding to specific regions of human C5orf22 (e.g., aa179-228). These peptides are conjugated to carrier proteins, injected into host animals (commonly rabbits), and the resulting antibodies are purified through immunoaffinity methods .

• Recombinant protein immunization: Some antibodies are produced using larger recombinant fragments of the C5orf22 protein as immunogens. For example, antibody orb354187 was generated using recombinant human C5orf22 protein (101-420AA) .

Both methods produce polyclonal antibodies with different binding characteristics and applications.

What methods should be used to validate C5orf22 antibody specificity?

For rigorous validation of C5orf22 antibodies, implement this multi-step approach:

• Genetic validation (gold standard): Test antibodies by immunoblot comparing parental cell lines to CRISPR/Cas9-generated C5orf22 knockout (KO) lines. Valid antibodies should show robust signal in parental cells that is absent or significantly reduced in KO cells .

• Orthogonal validation: Compare antibody-based detection with mRNA expression data or results from multiple antibodies targeting different epitopes of C5orf22 .

• Immunoprecipitation followed by mass spectrometry: This confirms the antibody's ability to enrich for C5orf22 and identifies potential cross-reacting proteins .

• Overexpression systems: Test antibody response to exogenously expressed tagged C5orf22 (e.g., Myc-DYKDDDDK tagged C5orf22) .

This comprehensive validation strategy ensures antibody specificity for the intended target and appropriate application .

How can I address the issue of non-specific binding with C5orf22 antibodies?

To reduce non-specific binding of C5orf22 antibodies:

• Optimize blocking conditions: Test different blocking agents (5% BSA, 5% non-fat milk, commercial blockers) to identify optimal blocking for your specific sample type and antibody.

• Titrate antibody concentration: Perform dilution series experiments to identify the minimum concentration that provides specific signal. For C5orf22 antibodies, recommended dilutions typically range from 1:200-1:500 for IHC and 1:250-1:2000 for WB, but optimization is necessary for each experimental system .

• Modify washing protocols: Implement more stringent washing procedures, including increased wash duration, volume, or detergent concentration.

• Include appropriate controls: Always include negative controls (C5orf22 knockout or knockdown samples) to distinguish between specific and non-specific signals .

• Pre-adsorption: Consider pre-adsorbing the antibody with the immunizing peptide to confirm specificity of observed signals.

Why is knockout validation critical for C5orf22 antibody assessment?

Knockout validation represents the most rigorous method for antibody specificity determination for several reasons:

• Unbiased validation: CRISPR/Cas9-mediated knockout provides an isogenic control lacking only the target antigen, allowing direct assessment of antibody specificity against the endogenous protein .

• Application versatility: Unlike other validation methods, genetic approaches are suitable for validating antibodies across all applications (WB, IHC, IF, etc.) .

• Historical limitations overcome: Previous antibody validation was hampered by lack of suitable controls, but modern gene editing techniques now make knockout-based validation feasible and accessible .

• Reveals problematic antibodies: Studies with C9ORF72 antibodies demonstrated that some widely used antibodies failed knockout validation despite being used in highly cited papers, raising concerns about previously reported findings. This same principle applies to C5orf22 antibody research .

For C5orf22 antibodies, implementation of knockout validation helps prevent publication of potentially misleading results based on non-specific antibody reactions.

What are the optimal conditions for using C5orf22 antibodies in Western blotting?

For optimal Western blot results with C5orf22 antibodies:

• Sample preparation:

  • Use RIPA or 1% Triton X-100 containing buffers for efficient protein extraction

  • Load 50μg of total protein per lane

  • Use 5-16% gradient gels for optimal resolution of C5orf22 (~49 kDa)

• Transfer conditions:

  • Transfer to nitrocellulose membranes (preferred over PVDF for C5orf22)

  • Verify transfer efficiency with Ponceau S staining

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBS-T (recommended for most C5orf22 antibodies)

  • Dilute primary antibodies according to manufacturer recommendations:

    • HPA043062: 0.04-0.4 μg/mL

    • orb587086: 1:500-1:2000

    • LS-C170545: 1:500-1:1000

  • Incubate overnight at 4°C for optimal results

• Detection:

  • For quantitative analysis, use fluorescently-labeled secondary antibodies and imaging systems like LI-COR Odyssey

  • For enhanced sensitivity, use HRP-conjugated secondaries with enhanced chemiluminescence

• Controls:

  • Include positive control (cell line with confirmed C5orf22 expression)

  • Include negative control (C5orf22 knockout or knockdown)

This methodology has been optimized based on published protocols for C5orf22 and related protein research.

How should I select the appropriate C5orf22 antibody for immunohistochemistry (IHC)?

For selecting the optimal C5orf22 antibody for IHC applications:

• Validation status: Prioritize antibodies like HPA043062 that have been validated by the Human Protein Atlas project through extensive tissue microarray testing on 44 normal human tissues and 20 common cancer types .

• Epitope considerations: Select antibodies targeting epitopes that remain accessible after fixation and antigen retrieval. For C5orf22, antibodies targeting the middle region (e.g., AA 179-228) often perform well in IHC applications .

• Fixation compatibility: Confirm the antibody is compatible with your fixation method (formalin-fixed paraffin-embedded vs. frozen sections). Some C5orf22 antibodies like those marked for "IHC (fro)" and "IHC (p)" are validated for both frozen and paraffin sections .

• Visualization format: For brightfield microscopy, use unconjugated primary antibodies with appropriate secondary detection systems. For fluorescence applications, consider directly conjugated antibodies like NBP2-98180AF700 (Alexa Fluor® 700 conjugated) .

• Dilution optimization: Always optimize antibody dilution for your specific tissue and conditions. For C5orf22 antibodies, recommended starting dilutions are typically 1:200-1:500 for IHC .

What cell lines are recommended as positive controls for C5orf22 antibody validation?

Based on protein expression databases and experimental evidence, these cell lines serve as appropriate positive controls for C5orf22 antibody testing:

• HEK-293 cells: Human embryonic kidney cells show detectable expression of C5orf22 and have been used successfully for recombinant C5orf22 expression, making them suitable for both endogenous and overexpression studies .

• MCF7 cells: Human breast cancer cell line with documented C5orf22 expression, used for Western blot validation of some commercial C5orf22 antibodies .

• PaxDB-recommended lines: Reference the PaxDB proteomics database to identify cell lines with high C5orf22 expression, following the approach used for antibody validation pipelines .

When selecting positive control cell lines, consider:

• Expression level of the target protein

• Compatibility with your experimental system

• Availability of isogenic knockout cells for validation

• Cell type relevance to your research question

As shown in antibody validation studies, using cell lines with relatively high target protein expression improves sensitivity and specificity assessment .

How can I troubleshoot weak or absent signal when using C5orf22 antibodies?

When facing weak or absent signal with C5orf22 antibodies, systematically address these potential issues:

• Protein expression levels:

  • Confirm C5orf22 expression in your sample using proteomics databases or RT-PCR

  • Consider using cell lines with known higher expression of C5orf22

  • For very low abundance, implement enrichment strategies (e.g., immunoprecipitation before detection)

• Antibody selection issues:

  • Test alternative C5orf22 antibodies targeting different epitopes

  • Verify antibody functionality using positive controls

  • Check antibody storage and handling conditions (avoid repeated freeze-thaw cycles)

• Protocol optimization:

  • Increase antibody concentration (within manufacturer's recommended range)

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

  • Optimize antigen retrieval methods for IHC/IF

  • For Western blots, try different protein extraction buffers to improve solubilization

• Detection system limitations:

  • Use more sensitive detection systems (e.g., enhanced chemiluminescence for WB)

  • Consider signal amplification techniques (e.g., tyramide signal amplification for IHC)

  • For fluorescent applications, select fluorophores with minimal background in your sample type

• Sample preparation concerns:

  • Ensure protein denaturation is complete for WB applications

  • Add protease inhibitors to prevent protein degradation

  • Check protein extraction efficiency with positive control proteins

Implementing this systematic approach will help identify and address specific causes of weak signal when working with C5orf22 antibodies.

What strategies can help resolve high background issues with C5orf22 antibodies in immunofluorescence?

To reduce high background when using C5orf22 antibodies in immunofluorescence applications:

• Fixation and permeabilization optimization:

  • Test different fixation methods (4% PFA, methanol, or acetone)

  • Adjust permeabilization conditions (concentration and duration of detergent exposure)

  • Include a quenching step to reduce autofluorescence (0.1-1% sodium borohydride or 50mM NH₄Cl)

• Blocking improvements:

  • Extend blocking time (1-2 hours at room temperature)

  • Test different blocking agents (normal serum from secondary antibody host species, BSA, casein, commercial blockers)

  • Add 0.1-0.3% Triton X-100 to blocking buffer to reduce non-specific membrane binding

• Antibody dilution and incubation:

  • Use more dilute antibody solutions (start with 1:500-1:1000 for C5orf22 antibodies)

  • Perform antibody dilutions in blocking buffer rather than plain buffer

  • For C5orf22 antibodies validated for IF applications, adhere to recommended dilutions (e.g., 0.25-2 μg/mL for HPA043062)

• Washing protocol enhancements:

  • Increase number and duration of wash steps

  • Use PBS-T (PBS + 0.1% Tween-20) for more effective washing

  • Perform final washes with plain PBS to remove detergent

• Controls and counterstaining:

  • Include secondary-only controls to assess non-specific secondary binding

  • Use appropriate counterstains that don't interfere with your signal

  • Consider using directly conjugated antibodies to eliminate secondary antibody background

These optimizations should significantly improve signal-to-noise ratio when working with C5orf22 antibodies in immunofluorescence applications.

How can I determine if inconsistent results with C5orf22 antibodies stem from technical variation or biological heterogeneity?

To distinguish between technical variation and biological heterogeneity when working with C5orf22 antibodies:

• Implement comprehensive controls:

  • Positive controls (samples with known C5orf22 expression)

  • Negative controls (C5orf22 knockout samples)

  • Internal controls (detection of housekeeping proteins in the same samples)

  • Technical replicates (repeated analysis of the same sample)

  • Biological replicates (multiple independent samples from the same condition)

• Quantitative assessment:

  • Use quantitative detection methods (e.g., LI-COR Odyssey system for WB)

  • Calculate coefficients of variation for technical replicates (<15% typically indicates acceptable technical reproducibility)

  • Apply appropriate statistical tests to determine significance of observed differences

• Multi-antibody approach:

  • Test multiple antibodies targeting different C5orf22 epitopes

  • Compare results from antibodies from different manufacturers/sources

  • Assess correlation between different antibody results (high correlation suggests biological signal rather than technical artifacts)

• Orthogonal validation:

  • Compare antibody-based results with mRNA expression data

  • Validate key findings with non-antibody methods (e.g., mass spectrometry)

  • Consider using tagged C5orf22 constructs for expression validation

• Documentation and standardization:

  • Maintain detailed records of all experimental conditions

  • Standardize protocols to minimize technical variation

  • Document antibody lot numbers, as lot-to-lot variation can significantly impact results

This systematic approach helps differentiate between technical noise and genuine biological variation in C5orf22 detection.

How can C5orf22 antibodies be implemented for co-immunoprecipitation to identify interaction partners?

For successful co-immunoprecipitation (co-IP) experiments to identify C5orf22 interacting partners:

• Antibody selection for co-IP:

  • Based on immunoprecipitation validation strategies used for similar proteins like C9ORF72, select antibodies that effectively precipitate endogenous C5orf22

  • Pre-test candidate antibodies by performing small-scale IPs followed by Western blot to assess enrichment efficiency

  • Identify antibodies that can capture at least 50-70% of C5orf22 from lysates for optimal results

• Buffer optimization:

  • Use mild lysis conditions to preserve protein-protein interactions (e.g., 1% NP-40 or 0.5% CHAPS buffer)

  • Include protease and phosphatase inhibitors

  • Consider crosslinking agents for transient interactions

  • Test multiple buffer conditions to optimize interaction preservation while maintaining antibody binding

• Experimental procedure:

  • Pre-couple antibodies to protein A/G beads (1-5 μg antibody per IP)

  • Prepare detergent-solubilized lysates (1-2 mg total protein per IP)

  • Include appropriate controls:

    • Beads alone (no antibody)

    • Isotype control antibody

    • Lysate from C5orf22 knockout cells

• Interaction analysis:

  • Perform mass spectrometry analysis of immunoprecipitates to identify potential binding partners

  • Confirm interactions by reciprocal co-IP or other interaction methods

  • Validate specificity by comparing proteins identified in C5orf22 IP versus control IPs

  • Focus on proteins enriched in C5orf22 IP from wild-type cells but absent in IPs from knockout cells

• Result interpretation:

  • Prioritize proteins consistently identified across technical replicates

  • Consider functional categories and pathway analysis of identified partners

  • Compare partners to known interactors of proteins with similar subcellular localization or function

This approach mirrors successful strategies used to identify interaction partners for other uncharacterized proteins .

What considerations are important when selecting C5orf22 antibodies for quantitative analysis of expression levels across different tissues or cell types?

For accurate quantitative analysis of C5orf22 expression across tissues or cell types:

• Antibody validation requirements:

  • Select antibodies validated in knockout/knockdown systems to ensure specificity

  • Verify linear detection range using titrated recombinant C5orf22 proteins

  • Confirm equal detection efficiency across different sample types

  • Assess potential interference from tissue-specific post-translational modifications

• Technical approach selection:

  • For tissue analysis: Choose between IHC with digital image analysis or Western blotting of tissue lysates

  • For cell-type specific analysis: Consider flow cytometry (if surface exposed) or single-cell imaging approaches

  • For absolute quantification: Implement approaches using recombinant C5orf22 protein standards

• Quantitative detection optimization:

  • For Western blotting: Use fluorescently-labeled secondary antibodies and imaging systems like LI-COR Odyssey

  • For IHC: Apply consistent staining protocols and calibrated image acquisition settings

  • Include standard curves when possible

  • Process all samples in parallel to minimize batch effects

• Normalization strategy:

  • Select appropriate housekeeping proteins as loading controls

  • Consider using total protein normalization (e.g., Ponceau S staining)

  • For tissue analysis, normalize to tissue-specific reference proteins rather than ubiquitous housekeeping proteins

  • Validate normalization approach for each tissue/cell type

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Account for biological variability between replicates

  • Consider multiple testing correction for comparisons across many tissues/cell types

  • Present data with appropriate measures of central tendency and dispersion

Following these guidelines ensures reliable quantitative comparisons of C5orf22 expression across diverse biological samples.

How can active learning approaches be integrated with C5orf22 antibody research to improve experimental efficiency?

Active learning strategies can significantly enhance C5orf22 antibody research efficiency:

• Epitope selection optimization:

  • Apply active learning algorithms to predict optimal antigenic regions of C5orf22 for antibody development

  • Iteratively test and refine epitope selection based on antibody performance feedback

  • This approach can reduce the number of required antigen variants by up to 35% compared to traditional methods

• Iterative antibody validation pipeline:

  • Start with a small set of validation experiments on candidate antibodies

  • Use results to inform which additional experiments would be most informative

  • Prioritize subsequent testing based on preliminary specificity assessments

  • This reduces resource expenditure on extensively validating poor candidates

• Application-specific optimization:

  • Begin with base protocol conditions

  • Design intelligent sampling of parameter space rather than exhaustive grid searches

  • Use Bayesian optimization to suggest next experimental conditions based on previous results

  • This approaches optimal conditions more rapidly than traditional one-factor-at-a-time optimization

• Cross-reactivity assessment:

  • Apply active learning to efficiently identify potential cross-reactive proteins

  • Test against predicted cross-reactive targets rather than random proteins

  • Reduce testing burden while maintaining confidence in specificity assessments

• Library-on-library screening approaches:

  • For developing new C5orf22 antibodies, implement active learning strategies to guide antibody-antigen pairing experiments

  • This can speed up the learning process by approximately 28 steps compared to random sampling approaches

  • Focus resources on the most informative experiments to rapidly converge on optimal antibodies

By integrating these active learning approaches, researchers can achieve more efficient and effective results in C5orf22 antibody development and application while minimizing experimental resources.

How can multiple antibody-based techniques be combined to provide comprehensive insights into C5orf22 biology?

A multi-technique approach using C5orf22 antibodies provides complementary insights:

• Integrated workflow example:

  • Begin with Western blotting to confirm expression and molecular weight in tissues/cells of interest

  • Follow with immunofluorescence to determine subcellular localization

  • Perform IHC to examine tissue distribution patterns

  • Use immunoprecipitation-mass spectrometry to identify interaction partners

  • Apply proximity ligation assays to confirm specific interactions in situ

• Technique complementarity benefits:

  • Biochemical techniques (WB, IP) provide quantitative data about expression levels and interactions

  • Imaging techniques (IF, IHC) provide spatial information about distribution and co-localization

  • Functional assays (e.g., antibody-mediated inhibition) connect molecular presence to biological activity

• Validation through cross-technique confirmation:

  • Verify C5orf22 subcellular localization through both biochemical fractionation+WB and immunofluorescence

  • Confirm protein-protein interactions through both co-IP and proximity ligation assays

  • Compare tissue expression patterns between IHC and tissue Western blots

• Data integration strategy:

  • Develop unified models incorporating expression, localization, and interaction data

  • Use bioinformatic approaches to integrate antibody-derived data with transcriptomic and proteomic datasets

  • Identify discrepancies between techniques as opportunities for biological discovery

This comprehensive approach has demonstrated success in characterizing previously uncharacterized proteins like C9ORF72, revealing its localization to phagosomes/lysosomes .

What advanced approaches can overcome limitations in traditional C5orf22 antibody applications?

To address limitations in conventional C5orf22 antibody applications:

• Proximity-dependent labeling approaches:

  • Express C5orf22 fused to BioID or TurboID to identify proximal proteins

  • This overcomes limitations in co-IP by capturing transient interactions and spatial proximity

  • Allows identification of the C5orf22 microenvironment without relying on stable interactions

• Single-molecule localization microscopy:

  • Apply super-resolution techniques (STORM, PALM) using validated C5orf22 antibodies

  • Achieve 10-20nm resolution compared to ~250nm in conventional microscopy

  • Provides precise nanoscale localization of C5orf22 relative to cellular structures

• Single-cell antibody-based proteomics:

  • Implement mass cytometry (CyTOF) using metal-conjugated C5orf22 antibodies

  • Measure C5orf22 expression alongside dozens of other proteins at single-cell resolution

  • Reveals cell-to-cell heterogeneity in expression and correlation with cellular states

• Spatially-resolved proteomics:

  • Apply imaging mass cytometry or multiplexed ion beam imaging (MIBI)

  • Maintain tissue architecture while measuring C5orf22 distribution

  • Correlate with numerous other proteins in the same tissue section

• Live-cell nanobody applications:

  • Develop nanobodies against C5orf22 that function in intracellular environments

  • Express as fusion proteins with fluorescent reporters for live-cell imaging

  • Track dynamics and trafficking of C5orf22 in real-time without fixation artifacts

These advanced approaches extend beyond limitations of traditional antibody applications while building upon the specificity verification principles established through rigorous antibody validation protocols .

How might emerging CRISPR-based technologies complement or replace traditional C5orf22 antibody applications?

CRISPR-based technologies are transforming protein detection and localization studies, offering alternatives and complements to traditional C5orf22 antibody approaches:

• Endogenous protein tagging:

  • Use CRISPR-Cas9 to knock-in fluorescent tags or epitope tags at the C5orf22 locus

  • Provides visualization or pulldown of C5orf22 at endogenous expression levels

  • Overcomes antibody specificity concerns while maintaining native regulation

  • Example advantage: Tagged C5orf22 can be tracked in live cells without fixation artifacts

• CUT&TAG and CUT&RUN technologies:

  • For studying C5orf22 DNA/chromatin interactions (if relevant)

  • Uses CRISPR-guided targeting rather than traditional ChIP-seq

  • Offers higher sensitivity and lower background than antibody-based ChIP approaches

  • Potentially applicable if C5orf22 has nuclear functions

• CRISPR interference/activation systems:

  • Deploy CRISPRi/a to modulate C5orf22 expression

  • Creates calibrated knockdown or overexpression systems for antibody validation

  • Generates graded expression levels to test antibody sensitivity and linearity

  • More precise than traditional siRNA approaches

• Programmable RNA-guided protein manipulation:

  • Adapt CRISPR systems to target C5orf22 protein directly

  • Enables acute protein degradation, relocalization, or modification

  • Provides temporal control not possible with genetic knockout approaches

  • Complements antibody detection by creating controlled experimental perturbations

• Integration with antibody-based approaches:

  • Use CRISPR knockout cells as definitive negative controls for antibody validation

  • Combine CRISPR-based perturbation with antibody-based detection for functional studies

  • Deploy CRISPR screens to identify genes affecting C5orf22 expression, localization, or function

  • Validate antibody-identified interactions through CRISPR perturbation of interaction partners