RNF39 Antibody

What is RNF39 and why is it relevant to research?

RNF39 (Ring Finger Protein 39) is a member of the RING finger protein family characterized by a zinc finger domain that facilitates protein-protein interactions. Recent research has implicated RNF39 as a potential biomarker in several pathological conditions, most notably in salivary duct carcinoma (SDC), where it forms part of a four-gene set (alongside ADAMTS1, DSC1, and IGLL5) for predicting aggressive disease progression. This gene set has emerging value as a predictive biomarker to stratify patients who may benefit from additional systemic or radiation therapies . RNF39's zinc finger domain suggests potential roles in transcriptional regulation, protein ubiquitination, and signal transduction pathways, making it an important target for fundamental research and clinical applications.

Which host species are commonly used for developing RNF39 antibodies?

RNF39 antibodies are primarily developed in several host species, each offering specific advantages for different research applications. Based on available commercial antibodies, the most common host species include:

Selection of the appropriate host species depends on your experimental design, particularly when conducting multi-labeling experiments where avoiding cross-reactivity is essential .

What applications are RNF39 antibodies validated for?

RNF39 antibodies have been validated for multiple experimental applications, with varying effectiveness depending on the specific antibody clone and format. The primary validated applications include:

• Western Blotting (WB): For detecting RNF39 protein in cell or tissue lysates

• Immunohistochemistry (IHC): For localization in tissue sections (both paraffin-embedded and frozen)

• Immunofluorescence (IF): For subcellular localization studies

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection

• Immunocytochemistry (ICC): For cellular localization studies

The application range varies by specific antibody product. For instance, monoclonal antibody clone 5E10 has been validated for WB, IHC, IF, and IHC(p), making it versatile for multiple experimental designs . When selecting an antibody, ensure it has been specifically validated for your intended application to avoid experimental failures.

How should I select between monoclonal and polyclonal RNF39 antibodies?

The choice between monoclonal and polyclonal RNF39 antibodies should be guided by your specific experimental requirements:

Monoclonal RNF39 Antibodies:

• Provide high specificity to a single epitope, reducing background

• Offer consistent lot-to-lot reproducibility for longitudinal studies

• Available clones such as 5E10 and 4D3 target specific regions of RNF39

• Best suited for applications requiring high specificity like identifying specific isoforms

Polyclonal RNF39 Antibodies:

• Recognize multiple epitopes, potentially increasing detection sensitivity

• Better for detecting proteins with post-translational modifications or in denatured states

• Available for various regions (N-terminal, C-terminal, and specific amino acid sequences)

• Advantageous for applications like IHC where antigen retrieval might alter epitopes

For critical research requiring absolute reproducibility across experiments, monoclonal antibodies like clone 5E10 offer consistent results. For maximum sensitivity in detecting low-abundance RNF39, polyclonal antibodies targeting multiple epitopes may provide better results .

What validation experiments should I perform before using an RNF39 antibody?

Before incorporating an RNF39 antibody into your research workflow, perform these validation experiments to ensure reliability:

• Positive and Negative Controls:

  • Use cell lines or tissues with known RNF39 expression levels

  • Include RNF39 knockout or knockdown samples as negative controls

• Epitope-Specific Validation:

  • For antibodies targeting specific RNF39 regions (like N-terminal EGRKAAKVNAGVGEKGIYTA sequence), confirm specificity with blocking peptides

  • Compare results from antibodies targeting different epitopes of RNF39

• Cross-Reactivity Assessment:

  • Evaluate potential cross-reactivity with other RING finger proteins, particularly in your species of interest

  • If working across species, verify the sequence homology of the target epitope (e.g., horse has 92% homology to human in some regions)

• Application-Specific Validation:

  • For WB: Confirm molecular weight (expected: approximately 45 kDa)

  • For IHC/IF: Compare staining patterns with published literature

  • For quantitative applications: Establish standard curves and determine linear detection range

• Antibody Titration:

  • Perform dilution series to identify optimal working concentration

  • Assess signal-to-noise ratio across dilutions

Complete validation ensures experimental reproducibility and prevents misleading results from non-specific antibody binding .

What are the optimal protocols for immunohistochemical detection of RNF39?

For effective immunohistochemical detection of RNF39 in tissue samples, follow this optimized protocol based on published methodologies:

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin following standard histological procedures

• Section tissues at 4-5 μm thickness onto positively charged slides

Antigen Retrieval:

• Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) for 20 minutes

• Alternative: EDTA buffer (pH 9.0) may provide better results for certain antibody clones

Immunostaining Protocol:

• Block endogenous peroxidase with 3% H₂O₂ in methanol (10 minutes)

• Block non-specific binding with 5% normal serum from same species as secondary antibody (30 minutes)

• Incubate with primary RNF39 antibody (recommended dilutions: 1:100 for monoclonal 5E10, 1:50-1:200 for polyclonal antibodies)

• Incubate overnight at 4°C or 1 hour at room temperature

• Apply appropriate HRP-conjugated secondary antibody

• Develop with DAB substrate and counterstain with hematoxylin

Controls and Validation:

• Include positive control tissues (based on the Human Protein Atlas data)

• Include negative controls (primary antibody omission and isotype controls)

This protocol has been successfully employed in salivary duct carcinoma research, where RNF39 protein expression was evaluated as a potential prognostic marker .

How can I optimize Western blotting conditions for RNF39 detection?

Successful Western blot detection of RNF39 requires careful optimization of several parameters:

Sample Preparation:

• Extract proteins using RIPA buffer supplemented with protease inhibitors

• Include phosphatase inhibitors if phosphorylated forms are of interest

• Determine optimal protein loading (typically 20-50 μg of total protein)

Gel Electrophoresis and Transfer:

• Use 10-12% polyacrylamide gels for optimal resolution of RNF39 (anticipated MW ~45 kDa)

• Transfer to PVDF membranes at 100V for 1 hour or 30V overnight at 4°C

• Verify transfer efficiency with reversible protein stains before blocking

Antibody Incubation:

• Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Dilute primary RNF39 antibody according to manufacturer's recommendations (typically 1:500-1:2000)

• Incubate with primary antibody overnight at 4°C with gentle agitation

• Wash thoroughly (4 × 5 minutes with TBST)

• Incubate with appropriate HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour

Detection and Troubleshooting:

• Use enhanced chemiluminescence (ECL) detection system

• Start with shorter exposure times (30 seconds) and increase as needed

• If background is high, increase washing steps or adjust blocking conditions

• If signal is weak, try longer primary antibody incubation or signal amplification systems

This optimized protocol incorporates best practices from published literature on RING finger protein detection and vendor recommendations for specific RNF39 antibodies .

Why might I observe non-specific binding with my RNF39 antibody?

Non-specific binding is a common challenge with antibody-based detection of RNF39. Several factors may contribute to this issue:

Causes of Non-Specific Binding:

• Structural Homology: RNF39 belongs to the RING finger protein family, which shares conserved domains with other family members. This structural similarity can lead to cross-reactivity, particularly with polyclonal antibodies.

• Antibody Quality: Some commercial antibodies may not undergo rigorous validation against a panel of similar proteins. Antibodies validated only against the immunogen peptide may show cross-reactivity in complex biological samples.

• Sample Complexity: Tissue samples, particularly those that are fixed and processed, can present epitopes that bind antibodies non-specifically due to protein modifications or denaturation.

Remediation Strategies:

• Epitope-Specific Antibodies: Choose antibodies targeting unique regions of RNF39. The N-terminal region has distinct sequences that can provide greater specificity .

• Blocking Optimization: Increase blocking agent concentration (5-10% normal serum or BSA) and duration (up to 2 hours at room temperature).

• Antibody Titration: Perform careful dilution series to identify the optimal concentration that maximizes specific signal while minimizing background.

• Pre-Absorption Controls: Pre-incubate your antibody with the immunizing peptide when available to confirm specificity of staining patterns.

• Alternative Validation: Confirm results using antibodies from different host species or targeting different epitopes of RNF39.

Implementing these strategies will significantly improve the signal-to-noise ratio in your RNF39 detection experiments .

How can I address inconsistent RNF39 detection in different tissue samples?

Inconsistent detection of RNF39 across tissue samples can result from both biological variation and technical factors:

Biological Factors:

• Expression Level Variability: RNF39 expression may naturally vary across different tissues, cell types, or disease states.

• Protein Localization: RNF39 may shuttle between subcellular compartments, affecting epitope accessibility.

• Post-Translational Modifications: Modifications may alter epitope recognition in tissue-specific ways.

Technical Solutions:

• Antigen Retrieval Optimization:

  • Test multiple antigen retrieval methods (heat vs. enzymatic)

  • Compare different buffer systems (citrate pH 6.0 vs. EDTA pH 9.0)

  • Adjust retrieval duration (10-30 minutes)

• Fixation Considerations:

  • Standardize fixation protocols across specimens

  • For prospective studies, consider using freshly frozen tissues in parallel

  • Document fixation duration for retrospective analyses

• Signal Amplification:

  • Implement tyramide signal amplification for low-abundance detection

  • Consider polymer-based detection systems for increased sensitivity

• Multi-Antibody Approach:

  • Use antibodies targeting different RNF39 epitopes in parallel

  • Compare results from monoclonal (e.g., clone 5E10) and polyclonal antibodies

• Quantitative Controls:

  • Include standardized positive control tissues in each experiment

  • Normalize signal intensity against housekeeping proteins

This comprehensive approach has proven effective in standardizing RNF39 detection in heterogeneous samples, such as those used in salivary duct carcinoma studies .

How is RNF39 antibody being utilized in cancer research?

RNF39 antibody has emerged as a valuable tool in cancer research, particularly in the study of salivary duct carcinoma (SDC) and potentially other malignancies:

Salivary Duct Carcinoma Research:
A groundbreaking study by Kohsaka et al. (2022) identified RNF39 as part of a four-gene prognostic signature (alongside ADAMTS1, DSC1, and IGLL5) for aggressive SDC. This research utilized RNF39 antibodies for immunohistochemical validation of expression patterns observed in genomic analyses .

The study demonstrated that:

• RNF39 protein expression correlates with specific transcriptional signatures

• Immunohistochemical detection of RNF39 can help stratify patients with different prognostic outcomes

• RNF39 protein levels may help predict response to adjuvant therapies

Methodological Approaches:

• Tissue Microarray Analysis: RNF39 antibodies are used to screen multiple patient samples simultaneously

• Correlation with Clinical Outcomes: Expression levels are analyzed in relation to patient survival and treatment response

• Multi-Marker Panels: RNF39 staining is often combined with other markers (CD3, ADAMTS1, DSC1) for improved prognostic value

Future Research Directions:

• Investigating RNF39's functional role in tumor progression through antibody-based protein interaction studies

• Developing RNF39-targeted therapeutics based on structural insights

• Expanding RNF39 biomarker validation to other cancer types with similar molecular features

These applications highlight the crucial role of high-quality, validated RNF39 antibodies in translational cancer research .

What are the challenges in studying RNF39 protein interactions using antibody-based approaches?

Investigating RNF39 protein interactions presents several unique challenges that require specialized antibody-based approaches:

Key Challenges:

• RING Domain Complexities:

  • The zinc finger RING domain of RNF39 mediates protein-protein interactions

  • These interactions may be transient or condition-dependent

  • Standard immunoprecipitation may disrupt weak or pH-sensitive interactions

• Post-Translational Modifications:

  • Ubiquitination activities associated with RING finger proteins may complicate detection

  • Modifications may mask epitopes recognized by available antibodies

• Conformational States:

  • RNF39 may adopt different conformations depending on binding partners

  • Some epitopes may be accessible only in specific conformational states

Advanced Methodological Solutions:

• Proximity Ligation Assays (PLA):

  • Use RNF39 antibodies in combination with antibodies against suspected interacting partners

  • PLA provides single-molecule resolution of protein interactions in situ

  • Protocol modifications: Use mild fixation (2% PFA, 10 minutes) and optimize antibody concentrations

• Cross-Linking Immunoprecipitation:

  • Implement protein cross-linking before cell lysis (1-2% formaldehyde, 10 minutes)

  • Use RNF39 antibodies conjugated to magnetic beads for gentle pull-down

  • Verify specificity with reciprocal immunoprecipitation using antibodies against interaction partners

• Antibody-Based Protein Complementation Assays:

  • Engineer split reporter proteins fused to anti-RNF39 antibody fragments

  • Reconstitution occurs when RNF39 interacts with tagged partner proteins

  • Provides dynamic, real-time monitoring of interactions

These advanced approaches can help overcome the inherent challenges in studying the dynamic protein interaction network of RNF39, providing insights into its functional roles in normal physiology and disease states .

How can multi-omics approaches incorporate RNF39 antibody data?

Integrating RNF39 antibody-based data into multi-omics research frameworks provides comprehensive insights into its biological functions and clinical relevance:

Integration Strategies:

• Proteogenomic Correlation:

  • Map RNF39 antibody-detected protein levels against RNA-seq expression data

  • Identify post-transcriptional regulation mechanisms

  • Correlation matrix example:

  Sample Type	RNF39 Protein (IHC H-score)	RNF39 mRNA (FPKM)	Correlation Coefficient
  Normal Tissue	10-50	5-20	0.72
  Tumor Tissue	80-200	30-120	0.63
  Cell Lines	40-180	15-90	0.81

• Functional Proteomics:

  • Use RNF39 antibodies for immunoprecipitation followed by mass spectrometry

  • Map the RNF39 interactome under different cellular conditions

  • Correlate with phosphoproteomics data to identify signaling networks

• Spatial Multi-Omics:

  • Combine RNF39 immunofluorescence with in situ transcriptomics

  • Map spatial distribution of RNF39 protein in relation to its transcriptional dependencies

  • Correlate with metabolomic profiles in tissue microenvironments

Implementation Protocol:

• Obtain tissue or cell samples and divide for parallel processing

• Process for antibody-based assays (IHC, IF, Western blot) and omics analyses (RNA-seq, proteomics)

• Perform RNF39 antibody validation on a subset of samples

• Establish normalization procedures across platforms

• Apply statistical integration methods (MOFA, DIABLO, mixOmics)

Case Study Application:
In salivary duct carcinoma research, RNF39 antibody data has been successfully integrated with transcriptomic profiles to validate a four-gene prognostic signature. This integration strengthened the clinical relevance of the findings and provided mechanistic insights into the role of RNF39 in disease progression .

Can the same RNF39 antibody be used across different species in comparative studies?

Cross-species application of RNF39 antibodies requires careful evaluation of epitope conservation and validation in each target species:

Epitope Conservation Analysis:
RNF39 sequence homology varies across species, with implications for antibody cross-reactivity. For example:

• Human-Horse homology: 92% in N-terminal regions

• Human-Mouse homology: Varies by region, highest in functional domains

• Human-Monkey homology: Generally high across the protein

Validated Cross-Species Applications:

Validation Protocol for Cross-Species Use:

• Perform sequence alignment of the antibody's target epitope across species of interest

• Test antibody on positive control tissues/cells from each species

• Confirm specificity using negative controls (knockout/knockdown where available)

• Optimize antibody concentration separately for each species

• Document species-specific banding patterns or staining characteristics

Practical Considerations:

• For evolutionary studies, consider using antibodies against the most conserved regions of RNF39

• For species-specific applications, select antibodies validated in your target organism

• When differences are observed across species, verify with multiple antibodies targeting different epitopes

This approach ensures reliable comparative studies while accounting for species-specific variations in RNF39 structure and expression .

What methodological modifications are needed when applying RNF39 antibodies to different model organisms?

Adapting RNF39 antibody protocols for different model organisms requires specific methodological adjustments:

Rodent Models (Mouse/Rat):

• Fixation: Reduce fixation time to 12-24 hours for smaller tissues

• Antigen retrieval: Increase retrieval time by 5-10 minutes

• Primary antibody: Use at 1.5-2× the concentration recommended for human tissues

• Background reduction: Include mouse-on-mouse blocking for mouse monoclonal antibodies on mouse tissues

• Validation: Compare with RNF39 knockout models when available

Non-Human Primates:

• Protocol modifications: Generally minimal due to high sequence homology with human RNF39

• Antibody selection: Monoclonal antibody 5E10 shows good cross-reactivity with monkey tissues

• Validation: Compare staining patterns with human tissues as reference

Large Animal Models (e.g., Horse):

• Tissue processing: Extend dehydration times for larger tissue blocks

• Antibody selection: N-terminal antibodies show 92% predicted reactivity with horse RNF39

• Optimization: Titrate antibody concentrations specifically for equine tissues

• Background reduction: Pre-absorb secondary antibodies with host species proteins

Model-Specific Western Blot Adjustments:

• Lysis buffer optimization: Species-specific protease inhibitor cocktails

• Loading control selection: Verify housekeeping protein conservation across species

• Expected banding patterns: Document species-specific molecular weight variations

These methodological modifications ensure optimal detection of RNF39 across different model organisms while maintaining experimental rigor and reproducibility .