RNF39 Antibody, HRP conjugated

What is RNF39 and what biological functions has it been associated with?

RNF39 (Ring Finger Protein 39) is encoded by a gene located within the major histocompatibility complex (MHC) class I region on chromosome 6. Studies suggest it plays a significant role in early phase synaptic plasticity and may be involved in maintaining long-term potentiation (LTP) . As a ring finger protein, it likely possesses E3 ubiquitin ligase activity, potentially participating in protein degradation pathways. Alternative splicing results in multiple transcript variants encoding different isoforms, adding complexity to its functional characterization . Recent research has also identified RNF39 as part of a four-gene signature associated with aggressive salivary duct carcinoma, suggesting potential roles in cancer progression .

What applications is the RNF39 antibody, HRP conjugated, optimized for?

The HRP-conjugated RNF39 antibody (ABIN7168263) is primarily optimized for enzyme-linked immunosorbent assay (ELISA) applications . The direct conjugation to HRP makes it particularly suitable for one-step detection systems, eliminating the need for secondary antibody incubation steps. While ELISA represents the validated application, researchers have successfully adapted similar HRP-conjugated antibodies for other techniques including immunohistochemistry with appropriate protocol modifications. The technical documentation emphasizes that optimal working dilutions should be determined experimentally by each investigator, indicating that performance optimization will be context-dependent . When considering alternative applications, researchers should conduct thorough validation studies to ensure specificity and sensitivity in their particular experimental systems.

What are the optimal conditions for using RNF39 antibody, HRP conjugated, in ELISA?

For optimal ELISA performance with HRP-conjugated RNF39 antibody, researchers should implement the following methodological approaches:

• Coating: Standard antigen coating at 1-10 μg/mL in carbonate/bicarbonate buffer (pH 9.6)

• Blocking: Use 1-5% BSA in PBS to minimize non-specific binding

• Antibody dilution: Begin with manufacturer's recommended dilution (specific to lot) and optimize through titration experiments

• Buffer compatibility: Maintain consistent pH (7.4) and avoid components that inhibit HRP activity

• Incubation: 1-2 hours at room temperature with gentle agitation

• Washing: Use PBS with 0.05% Tween-20, employing 4-5 thorough washes between steps

• Substrate selection: TMB (3,3',5,5'-Tetramethylbenzidine) provides good sensitivity and stable signal

• Signal development monitoring: Optimize development time (typically 5-30 minutes) based on signal-to-noise ratio

The antibody is supplied in a specific buffer containing 0.03% Proclin 300 as preservative and 50% glycerol, which should be considered when calculating final working concentrations . Each new lot of antibody should undergo validation testing to determine optimal working dilutions for the specific experimental system.

How can I validate the specificity of RNF39 antibody, HRP conjugated, in my experimental system?

Thorough validation of antibody specificity is essential for generating reliable results. For HRP-conjugated RNF39 antibody, implement the following validation strategy:

• Positive/negative controls: Compare signals between tissues/cells known to express high versus low/no RNF39

• Genetic validation: Test antibody performance in RNF39 knockdown/knockout samples versus wild-type controls

• Peptide competition: Pre-incubate antibody with excess immunizing peptide (AA 200-321) to confirm epitope specificity

• Cross-reactivity assessment: Test on samples from non-target species (antibody is human-specific)

• Molecular weight verification: Confirm detection of appropriate band size (~39 kDa) in Western blot analyses

• Comparative antibody validation: Compare results with alternative RNF39 antibodies targeting different epitopes

• Orthogonal validation: Correlate protein detection with mRNA expression levels via RT-qPCR

Implementing this comprehensive validation approach ensures confidence in experimental results and facilitates troubleshooting if inconsistencies arise.

What storage and handling protocols maximize the stability of RNF39 antibody, HRP conjugated?

Proper storage and handling are critical for maintaining antibody performance over time. For optimal stability of HRP-conjugated RNF39 antibody:

• Upon receipt: Aliquot into smaller volumes to minimize freeze-thaw cycles

• Storage temperature: Maintain at -20°C for long-term storage; -80°C may further extend shelf-life

• Working solution handling: Keep on ice during experiments and prepare fresh dilutions for each use

• Light exposure: Minimize exposure to light, as HRP conjugates can be photosensitive

• Buffer compatibility: Avoid sodium azide in working solutions, as it inhibits HRP activity

• Freeze-thaw cycles: Limit to absolute minimum, ideally no more than 3 cycles

• Thawing method: Thaw rapidly at room temperature followed by immediate transfer to ice

• Storage containers: Use amber or foil-wrapped tubes to protect from light

The datasheet specifies that the antibody is supplied in a buffer containing 0.03% Proclin 300 as preservative with 50% glycerol and 0.01M PBS at pH 7.4 . Proper adherence to these storage and handling guidelines will help maintain antibody performance throughout its shelf-life.

How does epitope location (AA 200-321) affect the utility of this antibody for detecting different RNF39 isoforms?

The epitope location (amino acids 200-321) has significant implications for the detection capabilities of this antibody across different RNF39 isoforms:

• Isoform coverage: The antibody can only detect isoforms that contain the AA 200-321 region intact

• Alternative splicing impact: If splice variants affect this region, certain isoforms may escape detection

• Domain structure relevance: This region may contain functional domains that influence protein interactions

• Post-translational modifications: Modifications within the epitope region may affect antibody binding affinity

• Protein conformation: The accessibility of this epitope may vary depending on protein folding and complex formation

Researchers should compare the epitope sequence against known RNF39 isoforms using sequence alignment tools to predict detection capabilities. When comprehensive isoform detection is required, using multiple antibodies targeting different epitopes is recommended for validation. The location within AA 200-321 may also provide advantages for detecting specific functionally-relevant domains of RNF39 .

What approaches can optimize RNF39 antibody, HRP conjugated, for applications beyond ELISA?

While primarily validated for ELISA, HRP-conjugated RNF39 antibody can be adapted for additional applications with appropriate optimization:

For Immunohistochemistry (IHC):

• Antigen retrieval: Optimize using heat-induced (citrate or EDTA buffer) or enzymatic methods

• Endogenous peroxidase quenching: 0.3% H₂O₂ treatment (10-30 minutes)

• Antibody dilution: Begin with 1:50-1:200 range and titrate for optimal signal-to-noise

• Incubation conditions: 1 hour at room temperature or overnight at 4°C in humidity chamber

• Signal amplification: Consider tyramide signal amplification for low-abundance targets

• Counterstaining: Use hematoxylin for nuclei visualization without interfering with HRP signal

For Western Blotting:

• Sample preparation: Use RIPA or NP-40 buffer with protease inhibitors

• Protein loading: Begin with 20-50 μg total protein

• Antibody dilution: Start with 1:500-1:1000 range

• Detection method: Use chemiluminescent HRP substrate with appropriate exposure optimization

• Membrane: PVDF may provide better protein retention than nitrocellulose

These adaptations require thorough validation and comparison with application-specific antibody formats to ensure reliable results .

What methodological considerations are important when using RNF39 antibody for studying major histocompatibility complex (MHC) class I-related processes?

RNF39's location within the MHC class I region on chromosome 6 raises specific methodological considerations for immunological research:

• Genetic background characterization: Document MHC haplotypes in experimental subjects/cell lines

• Linkage disequilibrium awareness: Consider co-inheritance patterns with other MHC genes

• Regulatory relationships: Investigate potential co-regulation between RNF39 and classical MHC molecules

• Immune stimulation experiments: Compare RNF39 expression under various immune activation conditions

• Cell-type specificity: Analyze expression patterns in antigen-presenting cells versus other cell types

• Multi-parameter analysis: Co-stain for RNF39 alongside MHC class I and other immune markers

For such studies, appropriate controls should include:

• Genetic controls: Matched MHC haplotype comparisons

• Treatment controls: Paired stimulated/unstimulated samples

• Cell type controls: Comparisons across relevant immune and non-immune cell populations

This systematic approach helps elucidate RNF39's potential roles in MHC-associated functions, particularly given its genomic context .

What is the significance of RNF39 in salivary duct carcinoma research?

Recent research has identified RNF39 as part of a clinically significant four-gene signature (ADAMTS1, DSC1, RNF39, and IGLL5) in salivary duct carcinoma (SDC) . This aggressive malignancy of major salivary glands has poor prognosis despite current treatment approaches. The research by Kohsaka S. et al. (2022) demonstrated several important aspects of RNF39's role:

• Prognostic value: RNF39 expression, as part of this gene signature, correlates with aggressive disease behavior

• Biomarker potential: The signature helps identify patients at high risk for recurrence

• Treatment stratification: May help select patients who would benefit from additional therapies

• Biological insights: Suggests involvement in processes related to inflammation and cell adhesion

• Translational relevance: Provides basis for developing immunohistochemical screening panels

For researchers studying SDC, the HRP-conjugated RNF39 antibody offers a valuable tool for validating genomic findings at the protein level through immunohistochemical analysis of patient samples. This application has direct clinical relevance for improving prognostication and treatment decision-making in this challenging malignancy .

How can RNF39 antibody, HRP conjugated, be utilized to study synaptic plasticity mechanisms?

The implication of RNF39 in synaptic plasticity and long-term potentiation maintenance opens several research applications:

• Expression analysis in neural tissues:

  • Quantify RNF39 protein levels in specific brain regions

  • Compare expression between developmental stages

  • Analyze changes following learning paradigms or LTP induction

• Subcellular localization studies:

  • Determine synaptic versus extrasynaptic distribution

  • Investigate translocation during plasticity events

  • Examine co-localization with synaptic markers

• Functional correlation studies:

  • Monitor RNF39 levels during electrophysiologically-confirmed LTP

  • Compare expression between potentiated and non-potentiated synapses

  • Correlate with memory formation in behavioral paradigms

• Methodological approach:

  • Sample preparation: Optimize protocols for synaptosome isolation or neuron-specific labeling

  • Stimulation protocols: Utilize established LTP induction paradigms (high-frequency stimulation, theta burst)

  • Controls: Include pathway-specific positive controls (known LTP-responsive proteins)

  • Quantification: Implement rigorous image analysis with appropriate normalization

This research direction could significantly advance understanding of the molecular mechanisms underlying synaptic plasticity and memory formation .

What factors might contribute to inconsistent results when using RNF39 antibody, HRP conjugated?

Several variables can impact experimental consistency when working with HRP-conjugated RNF39 antibody:

• Sample preparation factors:

  • Fixation variables (duration, temperature, fixative composition)

  • Protein extraction efficiency and denaturation completeness

  • Sample storage conditions affecting epitope integrity

• Antibody-related factors:

  • Freeze-thaw cycles degrading antibody quality

  • Lot-to-lot variability in manufacturing

  • Working dilution optimization

  • Secondary detection reagent compatibility

• Protocol-specific factors:

  • Blocking efficiency variations

  • Washing stringency differences

  • Incubation time/temperature inconsistencies

  • Substrate development kinetics

• Target-related factors:

  • Post-translational modifications affecting epitope availability

  • Protein-protein interactions masking binding sites

  • Expression level variations between samples

  • Epitope accessibility in different sample preparations

Implementing standardized protocols with detailed documentation of all variables facilitates troubleshooting when inconsistencies arise. Incorporation of consistent positive and negative controls across experiments provides essential quality benchmarks .

How can I troubleshoot high background issues with RNF39 antibody, HRP conjugated?

High background is a common challenge with HRP-conjugated antibodies. Implement this systematic troubleshooting approach:

• Blocking optimization:

  • Test alternative blocking agents (BSA, casein, normal serum)

  • Increase blocking duration (2-4 hours or overnight)

  • Add 0.1-0.3% Triton X-100 to blocking buffer for better penetration

• Antibody dilution refinement:

  • Perform careful titration with 2-fold serial dilutions

  • Pre-absorb antibody with relevant negative control tissue

  • Centrifuge diluted antibody before use (14,000 × g, 10 min) to remove aggregates

• Washing enhancements:

  • Increase number of washes (5-7 times)

  • Extend wash duration (10-15 minutes per wash)

  • Add 0.05-0.1% Tween-20 to washing buffers

• Endogenous enzyme inactivation:

  • Implement dual quenching (3% H₂O₂ followed by 0.1% sodium azide) for tissues

  • Add levamisole to block endogenous alkaline phosphatase

  • Use commercial background reducers specific for HRP systems

• Detection optimization:

  • Reduce substrate incubation time

  • Dilute substrate solution

  • Use alternative substrate with different sensitivity profile

Methodical modification of one variable at a time while maintaining others constant facilitates identification of the problematic step .

What are the critical considerations for designing quantitative experiments using RNF39 antibody, HRP conjugated?

Robust quantitative analysis requires careful experimental design incorporating these key elements:

• Standard curve implementation:

  • Include recombinant RNF39 protein standards

  • Prepare fresh standards for each experiment

  • Ensure standards span the full expected concentration range

• Replication strategy:

  • Minimum triplicate technical replicates

  • Independent biological replicates (n ≥ 3)

  • Include inter-assay calibrators across experimental runs

• Controls framework:

  • Positive controls (known RNF39-expressing samples)

  • Negative controls (confirmed RNF39-negative samples)

  • Background controls (no primary antibody)

  • Processing controls (identical treatment across all samples)

• Signal optimization:

  • Determine linear detection range through dilution series

  • Optimize exposure/development time for linearity

  • Validate signal specificity through competition experiments

• Normalization approach:

  • Total protein normalization methods (Ponceau, REVERT)

  • Housekeeping protein controls with validated stability

  • Consistent loading volumes and concentrations

• Data analysis:

  • Appropriate statistical tests based on data distribution

  • Multiple comparison corrections for complex experiments

  • Transparent reporting of outlier handling

This comprehensive approach ensures generation of reproducible, quantitatively reliable data suitable for publication-quality research .

How does the choice of detection substrate affect results when using RNF39 antibody, HRP conjugated?

The detection substrate significantly impacts sensitivity, signal stability, and dynamic range when working with HRP-conjugated RNF39 antibody:

Substrate selection should be guided by:

• Required detection threshold (based on anticipated RNF39 abundance)

• Dynamic range requirements of the experimental system

• Need for permanent vs. transient signal

• Available detection instrumentation

• Compatibility with downstream applications

Each substrate system requires specific optimization of development time and concentration for optimal signal-to-noise ratio .

What comparative factors should be considered when selecting between different RNF39 antibodies for specific applications?

When selecting the optimal RNF39 antibody for a specific application, researchers should evaluate these key comparative factors:

• Epitope considerations:

  • Epitope location (N-terminal, C-terminal, internal domains)

  • Conservation across species and isoforms

  • Accessibility in native vs. denatured states

• Host species and clonality:

  • Polyclonal (broader epitope recognition) vs. monoclonal (higher specificity)

  • Host species compatibility with experimental system

  • Available secondary detection systems

• Conjugation status:

  • Direct conjugates (HRP, fluorophores) for simplified workflows

  • Unconjugated for flexibility and signal amplification

• Validation documentation:

  • Extent of validation for specific applications

  • Species cross-reactivity testing

  • Knockout/knockdown validation data

• Application-specific performance:

  • Optimized for specific techniques (ELISA vs. IHC vs. WB)

  • Buffer compatibility with experimental system

  • Documented performance in relevant sample types

The search results reveal multiple RNF39 antibody options with varying characteristics:

• ABIN7168263: Rabbit polyclonal, HRP-conjugated, targeting AA 200-321, optimized for ELISA

• ABIN2774791: Rabbit polyclonal, unconjugated, N-terminal targeting, validated for WB and IHC

• ABIN1500723: Mouse monoclonal (clone 5E10), unconjugated, validated for WB, IHC, and IF

Selection should be guided by specific experimental requirements and validated through preliminary testing in the researcher's specific system.