RNF24 Antibody

What is RNF24 and what are its known functions?

RNF24 (Ring Finger Protein 24) is a 17 kDa protein encoded by the RNF24 gene located on chromosome 20 in humans . It contains a RING finger domain characteristic of many E3 ubiquitin ligases, though its precise enzymatic activity remains under investigation. Current research indicates that RNF24 may play an important role in TRPC (Transient Receptor Potential Canonical) intracellular trafficking . This suggests potential involvement in calcium signaling pathways and membrane protein regulation. The protein's relatively small size and its involvement in trafficking pathways make it an interesting target for studies of protein-protein interactions and cellular localization.

What types of RNF24 antibodies are currently available for research?

Several types of RNF24 antibodies are available for research applications, varying in host species, clonality, and conjugation:

Most commercially available antibodies are polyclonal, raised in rabbit hosts . The antibodies target different amino acid regions of RNF24, with common epitopes including AA 33-61 and AA 45-148 . This diversity allows researchers to select antibodies targeting different regions based on experimental requirements and protein accessibility in different applications.

What are the validated applications for RNF24 antibodies?

RNF24 antibodies have been validated for multiple experimental applications:

When selecting an RNF24 antibody, researchers should prioritize those validated for their specific application of interest. Cross-validation with multiple antibodies targeting different epitopes is recommended for confirming results, especially in novel experimental systems.

What is the species reactivity spectrum of available RNF24 antibodies?

The species reactivity of RNF24 antibodies varies by product, with most antibodies showing reactivity against human RNF24 and some also recognizing mouse and rat homologs:

When working with non-human models, sequence homology analysis between the target epitope and the corresponding region in the species of interest is recommended to predict cross-reactivity. Experimental validation is essential when using these antibodies in species not explicitly listed in the manufacturer's specifications.

What are the optimal storage conditions for RNF24 antibodies?

Proper storage is crucial for maintaining antibody performance. For RNF24 antibodies:

• Storage temperature: -20°C is standard for most products

• Buffer composition: Most are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3-7.4

• Stability: Typically stable for one year after shipment when stored properly

• Aliquoting: While recommended for many antibodies, some products specifically note that "aliquoting is unnecessary for -20°C storage"

To maximize shelf-life and performance, avoid repeated freeze-thaw cycles by preparing working aliquots upon first thaw. When working with conjugated antibodies (FITC, HRP, etc.), minimize exposure to light to prevent photobleaching of the fluorophore or degradation of the enzyme.

How should researchers validate the specificity of RNF24 antibodies?

Validation of antibody specificity is essential for generating reliable research data. For RNF24 antibodies, consider these validation approaches:

• Positive and negative control samples:

  • Positive controls: Cell lines or tissues known to express RNF24

  • Negative controls: RNF24 knockout samples or tissues with negligible expression

• Molecular weight verification:

  • Expected molecular weight: 17 kDa

  • Verify band size in Western blot applications

• Competing peptide blocking:

  • Pre-incubate antibody with immunizing peptide

  • Should abolish or significantly reduce signal

• Orthogonal method comparison:

  • Compare protein detection with RNA expression data

  • Use multiple antibodies targeting different epitopes

• Immunoprecipitation followed by mass spectrometry:

  • Ultimate confirmation of antibody target specificity

What are the recommended dilutions and incubation conditions for different applications?

Optimal working dilutions vary by application and specific antibody product:

Always perform dilution series experiments to determine optimal concentration for your specific experimental conditions. Signal-to-noise ratio and background levels should guide final dilution selection.

How can RNF24 antibodies be used to study protein-protein interactions?

RNF24 antibodies can facilitate the study of protein-protein interactions through several methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use RNF24 antibodies to pull down RNF24 and associated proteins

  • Western blot analysis of precipitated proteins can identify interaction partners

  • Critical controls include IgG control and reciprocal Co-IP

• Proximity Ligation Assay (PLA):

  • Combine RNF24 antibody with antibodies against potential interacting partners

  • PLA generates fluorescent spots only when proteins are in close proximity (<40 nm)

  • Particularly useful for studying RNF24's role in TRPC trafficking

• Immunofluorescence co-localization:

  • Use fluorescently conjugated RNF24 antibodies (e.g., FITC-conjugated)

  • Co-stain with antibodies against potential interacting proteins

  • Quantify co-localization using appropriate statistical methods

• FRET/BRET approaches:

  • Combine antibody-based detection with energy transfer techniques

  • Requires careful selection of fluorophore pairs and control experiments

When studying RNF24 interactions, researchers should consider its potential role in TRPC trafficking pathways and design experiments to capture both stable and transient interactions that may occur during the trafficking process.

What are the considerations for using RNF24 antibodies in cell-type specific studies?

When using RNF24 antibodies for cell-type specific analyses, researchers should consider:

• Expression pattern verification:

  • Confirm RNF24 expression in your cell type of interest using transcriptomic data

  • Use immunohistochemistry with cell-type specific markers for co-localization studies

• Fixation and permeabilization optimization:

  • Different cell types may require distinct fixation protocols

  • Test multiple conditions (PFA, methanol, acetone) to determine optimal preservation

  • Permeabilization conditions may need adjustment for different cellular compartments

• Background reduction strategies:

  • Employ appropriate blocking solutions specific to the cell type

  • Consider autofluorescence quenching for certain tissues/cell types

  • Use isotype controls matched to the host species and antibody class

• Signal amplification methods:

  • For low expression levels, consider tyramide signal amplification

  • Multiplex with cell-type specific markers for unambiguous identification

• Validation across independent samples:

  • Confirm staining patterns across multiple donors/animals

  • Account for potential inter-individual variation in expression levels

These considerations help ensure reliable and reproducible results when studying RNF24 expression and localization in specific cell populations.

How do recent advances in de novo antibody design impact RNF24 antibody development?

Recent advances in de novo antibody design have significant implications for developing improved RNF24 antibodies:

• Structure-based epitope targeting:

  • Computational approaches now enable precision targeting of specific epitopes

  • For RNF24, this could allow development of antibodies that distinguish between different functional states or conformations

  • Recent research demonstrates successful de novo antibody design across multiple target proteins

• Library construction strategies:

  • Modern techniques combine designed light chains (10² sequences) with designed heavy chains (10⁴ sequences) to create diverse libraries (10⁶ sequences)

  • This approach could generate RNF24 antibodies with enhanced specificity and reduced cross-reactivity

• Affinity and specificity engineering:

  • Computational design allows optimization of binding interfaces

  • May enable development of RNF24 antibodies capable of distinguishing between closely related protein subtypes or mutants

  • Potential for creating antibodies with comparable affinity to commercial antibodies but with improved specificity

• Yeast display technology integration:

  • De novo designed antibodies can be screened and validated using yeast display platforms

  • Facilitates rapid identification of high-affinity binders without requiring pre-existing antibody information

These advances hold promise for developing next-generation RNF24 antibodies with improved specificity, sensitivity, and functional capabilities that could enable more precise studies of RNF24 biology.

What are common issues when using RNF24 antibodies and how can they be resolved?

When troubleshooting RNF24 antibody experiments, systematic modification of one variable at a time will help identify the source of the problem. Maintaining detailed records of all experimental conditions facilitates troubleshooting and protocol optimization.

How can researchers optimize immunofluorescence protocols for RNF24 visualization?

For optimal immunofluorescence results with RNF24 antibodies:

• Fixation optimization:

  • Test different fixatives (4% PFA, methanol, acetone)

  • Fixation time may need adjustment (10-20 minutes) based on cell type

  • Consider mild fixation to preserve epitope accessibility

• Antigen retrieval considerations:

  • Heat-induced epitope retrieval may improve signal for some antibodies

  • Test citrate buffer (pH 6.0) and EDTA buffer (pH 8.0-9.0)

  • Enzymatic retrieval with proteinase K may help for certain applications

• Blocking and permeabilization:

  • Use 5-10% normal serum from the same species as secondary antibody

  • Test different permeabilization agents (0.1-0.5% Triton X-100, 0.1-0.5% Saponin)

  • Optimize permeabilization time (5-15 minutes) based on subcellular localization

• Antibody incubation:

  • For direct detection, FITC-conjugated RNF24 antibodies simplify protocols

  • For unconjugated primary antibodies, optimize both primary and secondary dilutions

  • Consider overnight incubation at 4°C for weak signals

• Signal amplification:

  • For low abundance proteins, consider tyramide signal amplification

  • Biotin-conjugated RNF24 antibodies with streptavidin-fluorophore detection can enhance sensitivity

• Counter-staining:

  • Include nuclear stain (DAPI/Hoechst) for orientation

  • Consider membrane or organelle markers based on expected RNF24 localization

  • Actin cytoskeleton staining provides cellular context

Detailed protocol records and systematic optimization are key to developing reproducible immunofluorescence methods for RNF24 visualization.

How should researchers interpret contradictory results from different RNF24 antibodies?

When faced with contradictory results from different RNF24 antibodies:

• Review epitope information:

  • Different antibodies target distinct regions (e.g., AA 33-61 vs. AA 45-148)

  • Epitope accessibility may vary in different experimental conditions or cellular contexts

  • Post-translational modifications may block specific epitopes

• Consider antibody specificity:

  • Polyclonal antibodies recognize multiple epitopes but may have higher cross-reactivity

  • Monoclonal antibodies have higher specificity but may miss protein variants

  • Validate specificity with appropriate controls (blocking peptides, knockdowns)

• Evaluate experimental conditions:

  • Different antibodies may perform optimally under different conditions

  • Fixation methods can dramatically affect epitope accessibility

  • Buffer compositions and blocking reagents can influence antibody binding

• Analyze protein state:

  • Some antibodies preferentially recognize native versus denatured protein

  • Protein complexes or interactions may mask certain epitopes

  • Protein conformation changes could expose or conceal epitopes

• Orthogonal validation approaches:

  • Confirm findings with non-antibody methods (e.g., GFP-tagging, mass spectrometry)

  • Use genetic approaches (knockdown/knockout) to verify specificity

  • Compare with mRNA expression data

• Consult literature and databases:

  • Review published validation data for specific antibodies

  • Check antibody validation databases for known issues

  • Consider reaching out to other researchers using these antibodies

Contradictory results may reflect genuine biological complexity rather than technical artifacts. Careful validation and comprehensive experimental design are essential for resolving such discrepancies.

How can RNF24 antibodies be integrated into multi-omics research frameworks?

Integration of RNF24 antibodies into multi-omics research enables comprehensive understanding of this protein's function:

• Proteomics integration:

  • Immunoprecipitation with RNF24 antibodies followed by mass spectrometry identifies interaction partners

  • Protein array analysis with RNF24 antibodies can reveal novel interactions

  • Comparison with proximity labeling approaches (BioID, APEX) provides complementary interaction data

• Genomics correlation:

  • ChIP-seq using RNF24 antibodies can identify potential DNA binding sites if RNF24 has nuclear functions

  • Correlate RNF24 protein levels with genomic/transcriptomic data to identify regulatory relationships

  • Integrate with GWAS data if RNF24 locus shows disease associations

• Transcriptomics correlation:

  • Combine RNF24 immunostaining with single-cell RNA-seq to correlate protein and mRNA levels

  • Identify cell populations with discordant protein/mRNA expression

  • Track changes in expression during development or disease progression

• Spatial omics applications:

  • Use RNF24 antibodies in multiplex imaging platforms (CODEX, MIBI, IMC)

  • Integrate with spatial transcriptomics for protein-RNA correlation at tissue level

  • Develop computational pipelines to analyze protein distribution patterns

• Functional proteomics:

  • Combine with ubiquitinome analysis to identify potential RNF24 substrates

  • Phospho-proteomics to identify signaling pathways affecting RNF24 function

  • Integrate with interactome databases to place RNF24 in functional networks

These integrated approaches provide a systems-level understanding of RNF24 biology beyond what can be achieved with single-method investigations.

What are the considerations for using RNF24 antibodies in high-throughput screening applications?

When employing RNF24 antibodies in high-throughput screening contexts:

• Assay miniaturization and automation:

  • Optimize antibody concentration for reduced volumes

  • Determine minimum incubation times without compromising signal

  • Validate antibody performance in automated liquid handling systems

• Signal detection optimization:

  • For fluorescence-based screening, FITC-conjugated RNF24 antibodies simplify workflows

  • HRP-conjugated antibodies provide amplification for chemiluminescent detection

  • Consider time-resolved fluorescence for improved signal-to-noise ratio

• Multiplexing considerations:

  • Test for antibody cross-reactivity in multiplex assays

  • Optimize antibody combinations for compatible detection systems

  • Consider antibody stripping protocols for sequential probing

• Quality control metrics:

  • Establish Z' factor for assay robustness assessment

  • Implement positive and negative controls on each plate

  • Monitor signal drift across plates and screening batches

• Data analysis pipelines:

  • Develop standardized analysis protocols for consistent interpretation

  • Implement machine learning for pattern recognition in complex phenotypes

  • Consider biological replicate strategies to minimize false positives/negatives

• Validation strategies:

  • Design secondary assays for hit confirmation

  • Include orthogonal approaches for mechanism validation

  • Consider dose-response testing for promising candidates

High-throughput applications require extensive initial optimization and quality control to ensure reliable and reproducible results across large sample sets.

How might RNF24 antibodies contribute to biomarker development or diagnostic applications?

While primarily research tools, RNF24 antibodies have potential applications in biomarker development:

• Expression pattern analysis:

  • Compare RNF24 expression across normal and disease tissues

  • Evaluate correlation with disease progression or patient outcomes

  • Assess value as part of multiplex biomarker panels

• Post-translational modification detection:

  • Develop modification-specific antibodies (phospho-RNF24, ubiquitinated RNF24)

  • Evaluate changes in modification patterns in disease states

  • Correlate with therapeutic response or resistance mechanisms

• Liquid biopsy applications:

  • Investigate RNF24 detection in circulating tumor cells or extracellular vesicles

  • Develop sensitive detection methods (e.g., proximity extension assays)

  • Evaluate prognostic or predictive value in longitudinal studies

• Imaging applications:

  • Explore potential for RNF24 antibodies in molecular imaging

  • Evaluate correlations between RNF24 localization and disease states

  • Consider companion diagnostic applications if therapeutically relevant

• Technical considerations for clinical translation:

  • Rigorous validation across multiple patient cohorts

  • Development of standardized protocols for clinical laboratory implementation

  • Robust quality control and reproducibility assessments

• Regulatory considerations:

  • Analytical validation requirements for clinical use

  • Clinical validation through appropriate trial design

  • Compliance with applicable regulatory frameworks

Any diagnostic application would require extensive validation beyond research use, including demonstration of clinical utility and analytical performance across diverse patient populations.

What are the most significant knowledge gaps in RNF24 biology that could be addressed with improved antibodies?

Despite available research tools, several key aspects of RNF24 biology remain underexplored:

• Enzymatic activity confirmation:

  • While RNF24 contains a RING finger domain characteristic of E3 ligases, its enzymatic activity and substrates remain poorly characterized

  • Improved antibodies could facilitate activity-based assays and substrate identification

• Tissue-specific functions:

  • Comprehensive analysis of RNF24 expression across tissues and cell types is incomplete

  • Cell type-specific roles and regulation mechanisms remain to be elucidated

• Subcellular localization dynamics:

  • While RNF24 may be involved in TRPC trafficking , its precise localization during different cellular processes is not fully mapped

  • Dynamic changes in response to stimuli or stress conditions are poorly understood

• Structure-function relationships:

  • How different domains contribute to RNF24 function

  • Conformational changes associated with activity or regulation

• Disease associations:

  • Potential roles in pathological processes remain largely unexplored

  • Connections to specific disease mechanisms or therapeutic opportunities

Next-generation antibodies with enhanced specificity, sensitivity, and functional capabilities could help address these knowledge gaps and advance understanding of RNF24 biology.

How might emerging antibody technologies improve RNF24 research in the coming years?

Emerging technologies will likely transform RNF24 antibody research:

• De novo design approaches:

  • Computational antibody design can create highly specific RNF24 binders

  • May enable development of conformation-specific antibodies

  • Could facilitate selective targeting of specific protein complexes

• Single-domain antibodies and nanobodies:

  • Smaller size enables access to sterically hindered epitopes

  • Improved penetration for imaging applications

  • Enhanced performance in certain applications like super-resolution microscopy

• Recombinant antibody technologies:

  • Improved consistency and reproducibility compared to animal-derived antibodies

  • Engineered modifications for specific applications

  • Potential for systematic epitope targeting across the entire RNF24 protein

• Spatially-resolved antibody applications:

  • Integration with spatial transcriptomics and proteomics

  • High-dimensional tissue analysis in health and disease contexts

  • Single-molecule localization methods for precise protein mapping

• Live-cell antibody applications:

  • Cell-permeable antibody formats for live-cell imaging

  • Intrabodies for tracking endogenous RNF24 in real-time

  • Optogenetic or chemogenetic antibody systems for functional perturbation