RNF19A Antibody, HRP conjugated

What is RNF19A and what are its key biological functions in experimental contexts?

RNF19A (Ring Finger Protein 19A, also known as Dorfin) is a member of the ring between ring fingers (RBR) protein family that functions as an E3 ubiquitin ligase. This protein contains two RING-finger motifs and an in-between RING fingers (IBR) motif at its N-terminus . RNF19A plays several critical roles in cellular function:

• Acts as a key regulator in protein quality control mechanisms through ubiquitin-mediated protein degradation pathways

• Functions as an oncogenic protein in non-small cell lung cancer (NSCLC), where it promotes cell proliferation and inhibits apoptosis

• Mediates K48-linked ubiquitination of target proteins such as TRAF6, thereby promoting their proteasomal degradation

• Interacts with p53 to regulate its stability through ubiquitin-mediated degradation in NSCLC cells

• May be involved in neuroinflammatory response regulation and has been implicated in neurodegenerative diseases including amyotrophic lateral sclerosis and Parkinson's disease

When designing experiments targeting RNF19A, researchers should consider its tissue-specific expression patterns and its various functional domains that mediate different protein-protein interactions.

What applications are most validated for RNF19A antibodies with HRP conjugation?

HRP-conjugated RNF19A antibodies have been validated for several research applications with specific recommended dilutions:

The primary advantages of HRP-conjugated antibodies in these applications include:

• Direct detection without requiring secondary antibodies, which simplifies experimental workflows

• Enhanced sensitivity due to enzymatic signal amplification properties of HRP

• Reduced background in multi-step detection protocols

• Compatibility with diverse substrates (colorimetric, chemiluminescent, or fluorescent)

When performing ELISA with HRP-conjugated RNF19A antibodies, researchers should optimize blocking conditions to minimize non-specific binding, as this remains the most common source of background signal.

How should researchers approach experimental validation of RNF19A antibody specificity?

Validating antibody specificity is critical for reliable experimental outcomes. For RNF19A antibodies, consider implementing these methodological approaches:

• Knockdown/knockout validation: Compare antibody signal between wild-type cells and those with RNF19A knockdown or knockout (using siRNA or CRISPR-Cas9). Search result #2 describes using siRNF19A (sequences: 5'-GATCCATTCTGAATTCCTA-3' and 5'-GCAAGTAGATATTGAGTCA-3') for effective knockdown validation .

• Cross-reactivity assessment: Test the antibody across multiple species to confirm expected reactivity patterns. The antibody in search result #1 shows reactivity with human and mouse RNF19A and predicted reactivity with pig, bovine, horse, sheep, rabbit, dog, and chicken samples .

• Epitope mapping: Verify that the antibody detects the expected molecular weight protein (approximately 90 kDa for RNF19A) .

• Positive control tissues: Use samples known to express RNF19A, such as NSCLC tissue samples or cell lines like A549, H292, H460, H661, H1299, and SK-MES-1, which show elevated RNF19A expression compared to normal lung epithelial cells .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (such as the synthetic peptide derived from human RNF19A amino acids 36-85) to confirm signal specificity .

What methodological considerations apply when using HRP-conjugated RNF19A antibodies for Western blotting?

When using HRP-conjugated RNF19A antibodies for Western blotting, researchers should implement these methodological approaches:

• Sample preparation optimization:

  • For cell lysates: Use NP-40 lysis solution containing protease inhibitors as described in study methodologies

  • Standardize protein loading (40 μg recommended) to ensure consistent results

• Blocking optimization:

  • Implement 5% skimmed milk blocking for 2 hours at 37°C to reduce non-specific binding

  • For problematic backgrounds, consider alternative blockers such as BSA

• Dilution testing:

  • For unconjugated RNF19A antibodies: 1:500-1:1000

  • For HRP-conjugated variants: 1:100-500

  • Perform serial dilutions to determine optimal signal-to-noise ratio

• Signal development considerations:

  • Use ECL detection reagents with appropriate exposure times

  • For weak signals, consider enhanced chemiluminescent substrates

  • Document using Bio-Imaging Systems for quantitative analysis

• Controls:

  • Include positive control lysates from Jurkat cells, which demonstrate detectable RNF19A expression

  • Include housekeeping protein controls (GAPDH recommended) as loading references

How can researchers effectively study RNF19A's role in ubiquitination pathways?

Investigating RNF19A's function in ubiquitination requires specific experimental approaches:

• Ubiquitination assays:

  • Co-transfect cells with relevant constructs (e.g., RNF19A and target proteins like TRAF6 or p53)

  • Detect K48-linked ubiquitination using specific antibodies

  • Analyze protein degradation through proteasome pathways

• Protein half-life determination:

  • Treat cells with cycloheximide (3 μg/ml) to inhibit protein synthesis

  • Collect samples at defined time points (0, 0.5, 1, 1.5, 2, and 2.5 hours)

  • Quantify protein degradation rates in presence/absence of RNF19A

• Interaction domain mapping:

  • RNF19A contains RING-finger motifs crucial for E3 ligase activity

  • Design truncation mutants to identify specific interaction domains

  • Use co-immunoprecipitation to confirm interactions with target proteins

• Ubiquitin chain specificity analysis:

  • RNF19A primarily mediates K48-linked ubiquitination, which targets proteins for proteasomal degradation

  • Use ubiquitin mutants (with specific lysine mutations) to confirm linkage types

  • Apply proteasome inhibitors (e.g., MG132) to validate degradation pathway involvement

What are the analytical approaches for studying RNF19A's role in oncogenic processes?

Based on research findings that RNF19A is overexpressed in NSCLC and promotes cell proliferation through p53 degradation, researchers should consider these analytical approaches:

• Clinical sample analysis:

  • Immunohistochemistry (IHC) protocols for tumor tissues:

    • Fix specimens in 10% formaldehyde, embed in paraffin, cut into 4 μm sections

    • Perform antigen retrieval using citrate solution (0.01 mol/L) under high temperature/pressure

    • Block endogenous peroxidase activity with 0.3% H₂O₂ for 20 minutes

    • Use anti-RNF19A antibody at 1:50 dilution (in 2% BSA) overnight at 4°C

• Expression correlation studies:

  • Analyze RNF19A expression in relation to clinical parameters:

  Clinical Parameter	Association with RNF19A	Statistical Significance
  Tumor size	Positive correlation	P < 0.05
  TNM stage	Positive correlation	P < 0.05
  Histological type	No significant correlation	P > 0.05
  Differentiation	No significant correlation	P > 0.05

• Functional studies in cell lines:

  • Proliferation assays following RNF19A knockdown or overexpression

  • Apoptosis assessment measuring markers like p53, p21, BAX, Cyclin D1, CDK4, CDK6, and BCL2

  • Colony formation assays to evaluate long-term growth effects

• Bioinformatic analyses:

  • Use databases like Oncomine (http://www.oncomine.org) with parameters:

    • Data type: mRNA

    • P-value < 0.01

    • Fold change > 1.5

  • Prognostic value assessment using GEPIA (http://gepia.cancer-pku.cn/)[2]

How should researchers interpret contradictory data regarding RNF19A function across different experimental models?

When encountering contradictory data about RNF19A function, researchers should implement these analytical approaches:

• Context-dependent function analysis:

  • RNF19A shows distinct roles in different cellular contexts:

    • Oncogenic in NSCLC through p53 degradation

    • Regulatory in immune responses by controlling TRAF6 degradation

    • Protective in neurodegenerative conditions

• Experimental model considerations:

  • Cell type-specific effects: Compare RNF19A expression across diverse cell types including:

    • Cancer cell lines (A549, H460)

    • Immune cells (THP-1)

    • Neuronal models

  • Species-specific differences: Human vs. mouse models may show divergent regulation patterns

• Interaction partner variability:

  • RNF19A functions through different binding partners:

    • NLRP11-dependent in immune contexts

    • p53-mediated in cancer models

  • Design co-immunoprecipitation experiments to identify context-specific interaction partners

• Expression level impact:

  • Analyze dose-dependent effects through titrated expression systems

  • Consider endogenous expression levels when interpreting overexpression studies

What methodological approaches can resolve specificity challenges with RNF19A antibodies in complex tissue samples?

When working with complex tissues, researchers face several specificity challenges that can be addressed through these methodological approaches:

• Epitope selection considerations:

  • N-terminal targeting antibodies: Suitable for detecting full-length RNF19A

  • C-terminal targeting antibodies: May detect specific isoforms

  • Internal epitope antibodies: Useful for detecting conserved domains

• Multiple detection methods implementation:

  • Combine immunoblotting with immunohistochemistry

  • Verify findings with mass spectrometry for unambiguous identification

  • Use ELISA for quantitative validation

• Pre-adsorption protocols:

  • Pre-incubate antibodies with the immunizing peptide

  • Gradually reduce antibody concentration to determine optimal specificity window

  • Document signal reduction patterns

• Isoform-specific detection strategies:

  • Design primers for RT-PCR validation of specific isoforms

  • Use antibodies targeting unique regions of particular isoforms

  • Correlate protein detection with transcript analysis

• Cross-reactivity mitigation:

  • Test reactivity across related proteins in the RBR family

  • Implement stringent washing protocols (recommended: wash thrice with TBST)

  • Optimize antibody dilutions based on tissue-specific background patterns

How can researchers effectively study the role of RNF19A in neuroinflammatory responses?

Recent research suggests RNF19A plays a role in neuroinflammation, particularly in the context of Japanese encephalitis virus (JEV) infection . To investigate this relationship, implement these methodological approaches:

• Epigenetic regulation analysis:

  • Study H3K27me3 modifications of the Rnf19a gene using:

    • Chromatin immunoprecipitation-sequencing (ChIP-seq)

    • Quantitative PCR of immunoprecipitated DNA

    • EZH2 inhibitor treatments to modulate histone methylation

• Microglial cell models:

  • Use BV2 microglial cell lines and primary mouse microglia

  • Analyze RNF19A expression under inflammatory conditions

  • Implement RNF19A knockdown and overexpression to assess functional impact

• Inflammatory marker assessment:

  • Measure pro-inflammatory cytokine production following RNF19A manipulation

  • Analyze signaling pathway activation (particularly RIG-I pathway components)

  • Correlate RNF19A expression with inflammatory marker levels

• In vivo models:

  • Study RNF19A expression in mouse brain during neuroinflammatory conditions

  • Implement cell-type specific knockdown to determine tissue-specific effects

  • Correlate pathological outcomes with RNF19A expression levels

• Mechanistic pathway analysis:

  • Investigate RNF19A's interaction with RIG-I and related signaling components

  • Assess ubiquitination patterns of inflammatory signaling proteins

  • Map degradation kinetics of key inflammatory mediators

What critical parameters should be optimized when developing immunoprecipitation protocols for studying RNF19A protein interactions?

Studying RNF19A's protein interactions requires carefully optimized immunoprecipitation protocols with these critical parameters:

• Lysis buffer composition optimization:

  • Use NP-40 lysis solution containing protease inhibitors as validated in published protocols

  • For detecting transient interactions, consider crosslinking approaches

  • Add deubiquitinase inhibitors (e.g., N-ethylmaleimide) when studying ubiquitination

• Antibody selection and validation:

  • Choose antibodies targeting specific domains:

    • For full-length protein interactions: N-terminal antibodies

    • For domain-specific interactions: Epitope-specific antibodies

• Bead selection and binding conditions:

  • For rabbit-derived RNF19A antibodies: Protein A-based matrices

  • Optimize binding time and temperature (typically 2-4 hours at 4°C)

  • Determine optimal antibody:lysate ratio through titration experiments

• Washing stringency determination:

  • Start with standard protocols (washing thrice with PBS)

  • Adjust salt concentration based on interaction strength

  • Optimize detergent levels to minimize non-specific binding

• Elution strategy selection:

  • For Western blot analysis: Direct boiling in sample buffer

  • For mass spectrometry: Peptide competition or low pH elution

  • For functional studies: Native elution conditions

• Controls implementation:

  • IgG control: Use species-matched IgG at equivalent concentration

  • Input control: Load 5-10% of starting material

  • Peptide competition: Pre-incubate antibody with immunizing peptide