RNF213 Antibody

What is RNF213 and why is it important to study with antibody-based techniques?

RNF213 (Ring Finger Protein 213) is a large E3 ubiquitin ligase (591 kDa) located in the cytosol and perinuclear space that contains both a C3HC4-type RING finger domain and six AAA+ ATPase domains, making it unique as the only known protein with both these functional domains. RNF213 is critical to study because it plays multifunctional roles in angiogenesis, lipid metabolism, and cell-autonomous immunity. The protein mediates ubiquitination of both proteins (e.g., FLNA and NFATC2) and lipids (e.g., bacterial lipopolysaccharide), indicating its involvement in diverse cellular processes. Additionally, RNF213 has been strongly associated with Moyamoya disease, a rare cerebrovascular disorder, making it an important target for investigating vascular pathologies .

For proper study of RNF213, antibody-based techniques are essential because they allow specific detection of this large protein in various experimental contexts including immunoblotting, immunohistochemistry, and immunofluorescence. These techniques enable researchers to observe native expression patterns, subcellular localization, protein-protein interactions, and changes in expression levels during disease states or in response to various stimuli.

What are the key considerations when selecting an RNF213 antibody for experimental purposes?

When selecting an RNF213 antibody for research, consider:

• Epitope location: Choose antibodies that target conserved regions of the protein, particularly if studying specific domains such as the RING finger or AAA+ ATPase domains.

• Validation status: Select antibodies validated for your specific application (Western blot, IHC, IF, IP). RNF213 antibodies have been successfully used in various applications including immunoblotting, immunohistochemistry, and immunofluorescence .

• Species reactivity: Ensure the antibody recognizes RNF213 in your experimental species. Human RNF213 has the UniProt ID Q63HN8 .

• Clonality: Polyclonal antibodies may offer higher sensitivity by recognizing multiple epitopes, while monoclonal antibodies provide higher specificity and consistency between batches.

• Size detection capability: Since RNF213 is a large protein (591 kDa), confirm the antibody can detect proteins in this high molecular weight range, which may require specialized gel systems and transfer protocols.

• Recognition of variants: Verify whether the antibody can detect known splice variants or mutant forms, especially when studying Moyamoya disease-associated variants.

• Background signal: Review literature or validation data to assess non-specific binding potential, which is particularly important when working with complex tissue samples.

• Published validation: Prioritize antibodies cited in peer-reviewed publications that demonstrate successful applications similar to your planned experiments.

What experimental controls should be included when using RNF213 antibodies?

When using RNF213 antibodies, the following controls are essential:

• Positive control: Include lysates from cells known to express RNF213 (such as endothelial cells or immune cells) to confirm antibody functionality.

• Negative control: Use appropriate RNF213-knockout or knockdown samples. Since complete knockout models may be challenging due to RNF213's role in development, siRNA or shRNA knockdown samples serve as good alternatives for antibody validation.

• Secondary-only control: Perform parallel experiments omitting the primary RNF213 antibody to identify any non-specific binding from the secondary antibody.

• Blocking peptide control: When available, pre-incubate the antibody with its immunizing peptide to demonstrate binding specificity.

• Loading control: Include detection of housekeeping proteins (β-actin, GAPDH, tubulin) to normalize RNF213 expression levels across samples.

• Titration series: Establish optimal antibody concentration by testing a range of dilutions to determine the concentration that provides the best signal-to-noise ratio.

• Cross-reactivity assessment: In multi-protein detection experiments, include controls to ensure the RNF213 antibody doesn't cross-react with other E3 ligases, especially those with similar RING finger domains.

• Isotype control: For immunoprecipitation or flow cytometry applications, include an isotype-matched irrelevant antibody control.

What are the optimal protocols for detecting RNF213 using Western blotting?

Optimized Western Blotting Protocol for RNF213 Detection:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors

  • Include 1-2 mM N-ethylmaleimide to preserve ubiquitination states

  • Sonicate briefly to shear DNA and reduce sample viscosity

  • Centrifuge at 14,000 × g for 15 minutes at 4°C to clear lysates

• Protein separation:

  • Use low percentage (4-6%) SDS-PAGE gels or gradient gels (4-12%) to effectively resolve the 591 kDa RNF213 protein

  • Load 50-100 μg of total protein per lane

  • Run at lower voltage (80-100V) for longer duration to improve separation of high molecular weight proteins

• Transfer:

  • Employ wet transfer systems with chilled transfer buffer containing 0.05-0.1% SDS to aid transfer of large proteins

  • Use PVDF membrane with 0.45 μm pore size

  • Transfer at 30V overnight at 4°C or use a high-molecular-weight transfer protocol

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk or BSA in TBST for 1-2 hours at room temperature

  • Incubate with primary RNF213 antibody (typically 1:500-1:2000 dilution) overnight at 4°C

  • Wash extensively with TBST (4-5 washes, 5-10 minutes each)

  • Incubate with secondary antibody (typically 1:5000-1:10000) for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) detection systems with extended exposure times

  • For more sensitive detection, consider using fluorescent secondary antibodies and imaging systems

• Special considerations:

  • Confirm complete transfer of high molecular weight proteins using reversible staining methods

  • Consider using a reference marker specifically for high molecular weight proteins

  • Optimize exposure time to avoid saturation while maintaining sensitivity

How can immunofluorescence with RNF213 antibodies be optimized for subcellular localization studies?

Optimized Immunofluorescence Protocol for RNF213 Subcellular Localization:

• Cell preparation:

  • Culture cells on glass coverslips or chamber slides coated with appropriate substrates

  • Consider using cells with known RNF213 expression (e.g., endothelial cells)

• Fixation options:

  • Primary method: 4% paraformaldehyde for 15 minutes at room temperature

  • Alternative method: Methanol fixation (100% methanol at -20°C for 10 minutes) may better preserve certain epitopes

  • Perform comparative analysis to determine optimal fixation for your specific RNF213 antibody

• Permeabilization:

  • Use 0.2% Triton X-100 in PBS for 10 minutes at room temperature

  • For gentler permeabilization, consider 0.1% saponin, which may better preserve membrane structures

• Blocking:

  • Block with 3-5% normal serum (matching the species of secondary antibody) with 0.1% BSA in PBS for 1 hour

• Antibody incubation:

  • Dilute primary RNF213 antibody (typically 1:100-1:500) in blocking buffer

  • Incubate overnight at 4°C in a humidified chamber

  • Wash 3-5 times with PBS

  • Incubate with fluorophore-conjugated secondary antibody (1:500-1:1000) for 1 hour at room temperature in the dark

• Co-staining considerations:

  • For co-localization studies, combine with markers for:

    • Perinuclear region: Nuclear envelope markers (Lamin A/C)

    • Cytosolic compartments: Specific organelle markers based on research question

    • For angiogenesis studies: Endothelial markers (CD31, VE-cadherin)

    • For immune function studies: Combine with bacterial or LPS markers

• Nuclear counterstaining:

  • Counterstain with DAPI or Hoechst (1:5000) for 5 minutes

  • Mount with anti-fade mounting medium

• Imaging recommendations:

  • Use confocal microscopy for precise subcellular localization

  • Capture Z-stacks to analyze the full cell volume

  • Include multi-channel imaging to assess co-localization with other cellular components

• Controls:

  • Include secondary-only control

  • Include RNF213 knockdown cells as negative control

RNF213 has been reported to localize primarily in the cytosol and perinuclear space , so particular attention should be paid to these regions when analyzing subcellular distribution patterns.

What are the recommended protocols for measuring RNF213 E3 ligase activity using antibody-based techniques?

Protocol for Measuring RNF213 E3 Ligase Activity:

• Immunoprecipitation-based ubiquitination assay:

  a) Cell lysate preparation:

  • Lyse cells in denaturing buffer (1% SDS, 50 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 mM DTT)

  • Heat at 95°C for 10 minutes to disrupt protein interactions

  • Dilute 10-fold with non-denaturing buffer (0.5% NP-40, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA)

  • Include protease inhibitors, phosphatase inhibitors, and deubiquitinase inhibitors (10 mM N-ethylmaleimide)

  b) Immunoprecipitation:

  • Pre-clear lysate with Protein A/G beads

  • Immunoprecipitate RNF213 using validated antibodies coupled to Protein A/G beads

  • Wash extensively with high-salt buffer followed by standard wash buffer

  c) Ubiquitination detection:

  • Analyze by SDS-PAGE and immunoblot

  • Probe with anti-ubiquitin antibody to detect ubiquitinated proteins

  • Re-probe with anti-RNF213 to confirm successful immunoprecipitation

• In vitro ubiquitination assay:

  a) Protein purification:

  • Purify recombinant RNF213 or immunoprecipitate from cell lysates

  • Verify purity by SDS-PAGE

  b) Ubiquitination reaction:

  • Combine purified RNF213 with E1 enzyme, E2 enzyme, ubiquitin, ATP, and substrate

  • For LPS ubiquitination: include purified LPS as substrate

  • For protein substrates: include recombinant FLNA or NFATC2

  • Incubate at 37°C for 1-2 hours

  c) Activity detection:

  • Analyze reaction products by SDS-PAGE and immunoblotting

  • Detect ubiquitinated products using anti-ubiquitin antibody

  • Alternatively, use fluorescently labeled ubiquitin for direct visualization

• Cellular ubiquitination assay:

  a) Cell preparation:

  • Transfect cells with tagged ubiquitin (HA-Ub or His-Ub)

  • Optionally co-transfect with RNF213 wild-type or mutant constructs

  • For bacterial studies, infect cells with appropriate pathogens (e.g., Salmonella)

  b) Enrichment of ubiquitinated proteins:

  • For His-Ub: perform Ni-NTA pulldown under denaturing conditions

  • For HA-Ub: perform anti-HA immunoprecipitation

  c) Detection:

  • Immunoblot with anti-substrate antibodies to detect ubiquitinated target proteins

  • For LPS ubiquitination: use anti-LPS antibodies

  • For known protein substrates: use antibodies against FLNA, NFATC2, etc.

• Controls and validation:

  • Include catalytically inactive RNF213 mutant (RING domain mutant)

  • Include reactions without ATP as negative control

  • For pathogen studies, include both wild-type bacteria and relevant bacterial mutants

How can RNF213 antibodies be applied to study Moyamoya disease mechanisms?

Methodological Approaches for Studying Moyamoya Disease Using RNF213 Antibodies:

• Patient-derived sample analysis:

  • Immunohistochemistry (IHC) on vascular tissue from MMD patients compared to controls

  • Western blot analysis of RNF213 expression levels in peripheral blood mononuclear cells (PBMCs) from patients with different RNF213 genotypes

  • Correlation of RNF213 protein levels with disease severity and progression

• Functional studies with disease-associated variants:

  • Generate cell lines expressing wild-type RNF213 or MMD-associated variants (especially p.R4810K)

  • Compare protein stability, subcellular localization, and E3 ligase activity between wild-type and mutant RNF213

  • Assess differences in substrate recognition and ubiquitination patterns

• Vascular remodeling investigation:

  • Use RNF213 antibodies in combination with endothelial markers in patient-derived induced pluripotent stem cells (iPSCs) differentiated into vascular cells

  • Track RNF213 expression during angiogenesis assays comparing wild-type and mutant conditions

  • Analyze RNF213's role in the non-canonical Wnt signaling pathway by monitoring ubiquitination and degradation of FLNA and NFATC2 during vessel regression

• Co-immunoprecipitation studies:

  • Identify RNF213-interacting proteins in vascular tissues from normal vs. MMD patients

  • Compare protein interaction networks between wild-type and mutant RNF213

  • Investigate differences in complex formation and substrate binding

• Imaging applications:

  • Use high-resolution confocal microscopy with RNF213 antibodies to analyze vascular smooth muscle cells and endothelial cells from patient samples

  • Implement proximity ligation assays to detect RNF213 interaction with potential binding partners in situ

  • Apply super-resolution microscopy techniques to analyze fine details of RNF213 localization in the perinuclear region

• Inflammatory response assessment:

  • Given RNF213's role in immunity, analyze inflammatory signaling pathways in patient-derived cells with MMD-associated mutations

  • Evaluate RNF213 expression in response to inflammatory stimuli in cells with wild-type vs. mutant RNF213

What approaches can be used to investigate RNF213's role in antimicrobial immunity using antibodies?

Methodological Approaches for Studying RNF213's Antimicrobial Function:

• Infection models:

  • Monitor RNF213 expression and localization during bacterial infection using immunofluorescence

  • Track recruitment of RNF213 to bacterial entry sites or cytosolic bacteria (particularly for Salmonella or Shigella)

  • Analyze co-localization with bacterial markers and autophagic machinery

• LPS ubiquitination detection:

  • Use co-immunoprecipitation with RNF213 antibodies followed by detection with anti-LPS antibodies

  • Implement proximity ligation assays to visualize RNF213-LPS interactions in situ

  • Develop a three-color immunofluorescence approach to simultaneously visualize RNF213, ubiquitin, and LPS

• Bacterial subversion mechanism studies:

  • Investigate how pathogens like Shigella target RNF213 through effectors like IpaH1.4

  • Compare RNF213 protein levels during infection with wild-type vs. effector-deficient bacteria

  • Analyze protein-protein interactions between bacterial effectors and RNF213 domains

• Antibacterial autophagy pathway:

  • Assess the role of RNF213 in antibacterial autophagy by monitoring LC3 recruitment to bacteria

  • Compare autophagy induction between wild-type and RNF213-depleted cells using confocal microscopy

  • Investigate the relationship between RNF213-mediated LPS ubiquitination and downstream autophagy activation

• LUBAC complex interaction:

  • Study RNF213's role in recruiting the LUBAC complex during infection

  • Use co-immunoprecipitation with RNF213 antibodies to detect LUBAC components

  • Analyze linear ubiquitin chain formation in the presence or absence of RNF213

• RNF213 expression regulation:

  • Analyze RNF213 protein levels in response to different microbial stimuli and interferon treatment

  • Compare expression patterns in different immune cell types using flow cytometry or immunohistochemistry

  • Investigate post-translational modifications of RNF213 during infection

How can RNF213 antibodies be used to investigate its role in lipid metabolism disorders?

Methodological Approaches for Studying RNF213's Function in Lipid Metabolism:

• Lipid droplet association studies:

  • Use co-immunofluorescence to analyze RNF213 localization relative to lipid droplets (stained with BODIPY or similar markers)

  • Implement super-resolution microscopy to determine precise spatial relationships

  • Examine changes in localization under different metabolic conditions (normal vs. high fat)

• Regulation of lipolysis:

  • Investigate how RNF213 inhibits the lipolytic process by analyzing interactions with lipolysis-related proteins

  • Use proximity ligation assays to detect in situ interactions between RNF213 and lipolytic enzymes

  • Compare lipid droplet dynamics in cells with normal vs. depleted RNF213 levels

• Fatty acid desaturation pathway:

  • Study RNF213's role in inhibiting desaturation of fatty acids

  • Identify potential targets in the desaturation pathway using co-immunoprecipitation with RNF213 antibodies

  • Analyze protein levels of desaturases in RNF213 wild-type vs. depleted conditions

• Lipotoxicity protection mechanisms:

  • Assess how RNF213 regulates lipotoxicity through inhibition of fatty acid desaturation

  • Analyze cell survival and ER stress markers in relation to RNF213 expression levels

  • Use immunoprecipitation to identify ubiquitinated targets in the lipid metabolic pathway

• Metabolic disease models:

  • Apply RNF213 antibodies in tissues from metabolic disease models (obesity, diabetes)

  • Compare RNF213 expression patterns between healthy and diseased states

  • Correlate RNF213 levels with lipid accumulation and metabolic parameters

How can researchers distinguish between specific RNF213 domains using domain-specific antibodies?

Methodological Approach for Domain-Specific RNF213 Analysis:

The large size of RNF213 (591 kDa) with its multiple functional domains presents unique opportunities for domain-specific investigations. RNF213 contains a RING-type zinc finger domain responsible for E3 ligase activity and six AAA+ ATPase domains . Domain-specific antibodies can provide insights into:

• Domain-specific antibody selection and validation:

  • Choose antibodies targeting specific epitopes within:

    • RING domain (for investigating E3 ligase function)

    • AAA+ ATPase domains (for investigating ATP-dependent functions)

    • RZ finger domain (important for LPS ubiquitination)

  • Validate specificity using:

    • Domain deletion constructs

    • Point mutants in specific domains

    • Peptide competition assays with domain-specific peptides

• Functional domain analysis applications:

  • Use domain-specific antibodies to:

    • Track conformational changes upon ATP binding/hydrolysis

    • Identify which domains interact with specific binding partners

    • Determine accessibility of domains in different cellular contexts

• Domain-specific Co-IP strategy:

  • Implement a strategy using domain-specific antibodies to:

    • Pull down RNF213 via different domains

    • Compare interactome differences when targeting different domains

    • Identify domain-specific post-translational modifications

• Structural biology applications:

  • Use domain-specific antibodies as tools for:

    • Cryo-EM studies to stabilize specific conformations

    • Domain accessibility mapping under different conditions

    • Epitope mapping to reveal structural features

• Disease-associated variant analysis:

  • Apply domain-specific antibodies to:

    • Determine how MMD-associated mutations affect specific domain functions

    • Compare domain accessibility between wild-type and mutant proteins

    • Analyze domain-specific interactions in patient-derived samples

What are the strategies for capturing transient RNF213-substrate interactions during ubiquitination reactions?

Advanced Methods for Detecting RNF213-Substrate Interactions:

Capturing the often transient interactions between E3 ligases and their substrates presents significant technical challenges, particularly for a large protein like RNF213. The following methodological approaches can help researchers overcome these challenges:

• Substrate trapping approaches:

  • Generate catalytically inactive RNF213 mutants (RING domain mutations) that can bind but not ubiquitinate substrates, prolonging the interaction

  • Use protein crosslinking methods (e.g., formaldehyde, DSP, or photo-crosslinkers) to stabilize transient interactions

  • Implement BioID or TurboID proximity labeling fused to RNF213 to identify proteins in close proximity

• Real-time interaction monitoring:

  • Develop FRET-based assays with fluorescently labeled RNF213 and potential substrates

  • Use bioluminescence resonance energy transfer (BRET) for live-cell monitoring of interactions

  • Implement single-molecule tracking microscopy to visualize interaction dynamics

• Modified immunoprecipitation strategies:

  • Perform tandem affinity purification with RNF213 antibodies under carefully optimized conditions

  • Use rapid immunoprecipitation techniques to capture short-lived complexes

  • Include ATP in buffers to stabilize AAA+ domain-dependent interactions

• In situ visualization techniques:

  • Apply proximity ligation assays (PLA) to visualize RNF213-substrate interactions in fixed cells

  • Implement split-GFP complementation assays for live-cell visualization

  • Use super-resolution microscopy techniques to detect co-localization at nanometer resolution

• Structural biology approaches:

  • Generate antibody fragments (Fabs) that can stabilize RNF213-substrate complexes for structural studies

  • Use cryo-electron microscopy with domain-specific antibodies to visualize substrate binding modes

  • Implement hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

How can researchers validate RNF213 antibody specificity in complex tissues and challenging experimental conditions?

Advanced Validation Strategies for RNF213 Antibodies:

Validating antibody specificity for a large, complex protein like RNF213 (591 kDa) presents significant challenges, particularly in diverse experimental conditions and tissue types. The following comprehensive approach ensures reliable detection:

• Genetic validation approaches:

  • Use CRISPR/Cas9-engineered RNF213 knockout cells as definitive negative controls

  • Implement siRNA/shRNA knockdown with titration to create a gradient of expression for sensitivity assessment

  • Generate epitope-tagged RNF213 knock-in cell lines for parallel detection with anti-tag and anti-RNF213 antibodies

  • For tissues, use conditional knockout models if complete knockout is not viable

• Biochemical validation methods:

  • Perform peptide competition assays with the immunizing peptide

  • Validate using recombinant protein fragments spanning different RNF213 domains

  • Implement multiple antibodies targeting different epitopes to confirm specificity

  • Use mass spectrometry to confirm the identity of immunoprecipitated proteins

• Tissue-specific validation:

  • Include tissue-specific controls (e.g., brain vasculature for Moyamoya studies)

  • Compare staining patterns across multiple tissue types with known RNF213 expression patterns

  • Employ RNA-protein correlation using parallel RNA-seq and protein detection

  • Use RNAscope or similar in situ hybridization techniques alongside immunohistochemistry

• Technical optimization for challenging conditions:

  • Systematically compare fixation methods (paraformaldehyde, methanol, acetone)

  • Test multiple antigen retrieval protocols for formalin-fixed tissues

  • Optimize signal amplification methods for low-abundance detection

  • Establish titration curves across different experimental conditions

• Advanced imaging validation:

  • Use super-resolution microscopy to confirm expected subcellular localization patterns

  • Implement spectral imaging to distinguish true signal from autofluorescence

  • Perform co-localization studies with known interaction partners

  • Use tissue clearing techniques for 3D validation in thick tissue samples

What are the common technical challenges when detecting RNF213 and how can they be overcome?

Common Challenges and Solutions for RNF213 Detection:

• High molecular weight detection issues:

  Challenge: RNF213's large size (591 kDa) makes it difficult to resolve and transfer in standard Western blotting protocols.

  Solutions:

  • Use low percentage gels (3-6%) or gradient gels (4-12%)

  • Implement specialized transfer techniques:

    • Extended transfer times (overnight at 30V)

    • Add 0.05% SDS to transfer buffer

    • Use discontinued or pulsed-field electrophoresis

  • Validate complete transfer with reversible staining methods

  • Consider using capillary-based Western systems for improved transfer of high MW proteins

• Low endogenous expression levels:

  Challenge: Detecting native RNF213 in many cell types can be difficult due to relatively low expression.

  Solutions:

  • Use signal enhancement methods:

    • HRP-conjugated polymers instead of standard secondary antibodies

    • Tyramide signal amplification for immunohistochemistry

    • Extended exposure times for Western blots

  • Concentrate protein by immunoprecipitation before analysis

  • Induce expression with interferon treatment before analysis

  • Identify cells with higher endogenous expression as positive controls

• Non-specific binding and background:

  Challenge: Large proteins often show non-specific binding patterns or cross-reactivity.

  Solutions:

  • Optimize blocking conditions:

    • Test different blocking agents (milk, BSA, normal serum)

    • Increase blocking time or concentration

  • Increase washing stringency:

    • More frequent washes

    • Higher salt concentration in wash buffers

  • Pre-adsorb antibodies with cell lysates from RNF213 knockout cells

  • Use monoclonal antibodies for higher specificity when available

• Fixation and epitope accessibility issues:

  Challenge: Different fixation methods can affect epitope accessibility.

  Solutions:

  • Compare multiple fixation methods:

    • Paraformaldehyde (2-4%)

    • Methanol/acetone

    • Glyoxal-based fixatives

  • Optimize antigen retrieval:

    • Heat-induced epitope retrieval

    • Enzymatic retrieval

    • pH-controlled buffers

  • Use permeabilization optimization:

    • Test different detergents (Triton X-100, saponin, digitonin)

    • Vary permeabilization time and concentration

• Post-translational modifications altering detection:

  Challenge: Ubiquitination or other modifications may mask epitopes or alter migration patterns.

  Solutions:

  • Include deubiquitinases inhibitors in lysis buffers

  • Test antibodies targeting different domains/epitopes

  • Use denaturing conditions in sample preparation

  • Consider phosphatase treatment to eliminate charge-based mobility shifts

How should researchers interpret conflicting data from different RNF213 antibodies?

Strategies for Resolving Conflicting RNF213 Antibody Data:

Researchers frequently encounter situations where different antibodies against RNF213 produce seemingly contradictory results. This comprehensive framework helps systematically address such discrepancies:

• Epitope mapping and comparison:

  • Determine the exact epitopes recognized by each antibody

  • Assess whether epitopes might be differentially affected by:

    • Protein conformation changes due to experimental conditions

    • Post-translational modifications masking specific regions

    • Domain-specific interactions with binding partners

  • Consider whether antibodies recognize different splice variants or isoforms of RNF213

• Validation hierarchy establishment:

  • Create a validation priority framework:

    • Assign higher confidence to results confirmed by genetic approaches (KO/KD)

    • Prioritize antibodies with extensive published validation

    • Consider monoclonal antibodies more reliable for specific applications

    • Weigh results from multiple detection methods (e.g., IF confirmed by WB)

  • Implement orthogonal detection methods to break ties between conflicting antibodies

• Technical parameter analysis:

  • Systematically evaluate whether discrepancies arise from:

    • Different fixation or extraction methods affecting epitope accessibility

    • Variations in antibody concentration or incubation conditions

    • Differential sensitivity to denaturation or native conditions

    • Batch-to-batch variability in polyclonal antibodies

  • Create standardized protocols to directly compare antibodies under identical conditions

• Application-specific optimization:

  • Recognize that different antibodies may perform optimally in different applications:

    • Some antibodies work well for Western blot but poorly for IF

    • Others may excel in native IP but fail in denaturing conditions

  • Develop application-specific validation criteria rather than expecting universal performance

• Biological context consideration:

  • Assess whether conflicting results reflect actual biological variability:

    • Cell type-specific post-translational modifications

    • Context-dependent protein interactions masking epitopes

    • Different subcellular pools of RNF213 with distinct properties

    • Disease-specific alterations in protein structure or localization

How can researchers differentiate between RNF213 and other RING finger proteins in complex samples?

Advanced Methods for Specific RNF213 Detection Among RING Proteins:

The human genome encodes hundreds of RING finger proteins with structural similarities to the RING domain of RNF213, creating potential specificity challenges. The following comprehensive approach ensures specific detection:

• Unique domain combination exploitation:

  • Target detection strategies to RNF213's unique combination of domains:

    • No other protein combines RING domains with six AAA+ ATPase domains

    • Design antibodies targeting the junction regions between these domains

    • Use the unique RZ finger domain involved in LPS ubiquitination for specific detection

  • Implement sandwich assays requiring recognition of multiple unique domains

• Size-based differentiation:

  • Leverage RNF213's exceptional size (591 kDa):

    • Use size exclusion chromatography before immunodetection

    • Implement specialized gel systems optimized for very large proteins

    • Apply gradient gels that clearly separate high molecular weight proteins

  • Combine molecular weight determination with specific epitope detection

• Sequence divergence targeting:

  • Focus antibody development on regions with lowest homology to other RING proteins:

    • Target non-conserved loops or linker regions

    • Avoid highly conserved zinc-coordinating residues in antibody design

    • Develop peptide-specific antibodies against unique RNF213 sequences

  • Validate specificity against closely related RING finger proteins

• Functional discrimination approaches:

  • Exploit RNF213's unique functional characteristics:

    • Analyze ATP-dependent activities not present in most RING proteins

    • Target RNF213's distinctive ability to ubiquitinate lipids like LPS

    • Focus on unique substrate specificities (FLNA, NFATC2)

  • Implement activity-based probes that selectively label active RNF213

• Advanced purification and detection:

  • Use tandem purification strategies:

    • Sequential immunoprecipitation with antibodies against different domains

    • Combine size-based separation with immunoaffinity purification

    • Implement substrate-based affinity capture for functional RNF213

  • Apply mass spectrometry for definitive identification after enrichment