RNF213 Antibody, Biotin conjugated

What is RNF213 and what is its biological significance?

RNF213 is a large multifunctional protein that serves as an E3 ubiquitin ligase, capable of catalyzing ubiquitination of both proteins and lipids. It contains a distinctive C3HC4-type RING finger domain that binds two zinc atoms and facilitates protein-protein interactions . RNF213 plays critical roles in various biological processes including lipid metabolism, angiogenesis, and cell-autonomous immunity . Most notably, RNF213 has been identified as the major susceptibility factor for Moyamoya disease, a progressive cerebrovascular disorder that can lead to brain stroke in both adults and children . Understanding RNF213's structure and function provides valuable insights into vascular development and maintenance, particularly in the context of cerebrovascular diseases.

What are the structural characteristics of RNF213?

RNF213 is an exceptionally large protein with a molecular weight of 584 kDa (3280 amino acids) . Cryo-EM structural analysis reveals that it adopts an intricate, intertwined fold organized into three major modules. The protein contains AAA ATPase domains, with only AAA3 and AAA4 being catalytically competent . RNF213 has several distinctive structural features:

• It contains RNF213-specific insertions that appear to have structural roles in stabilizing the AAA ring

• A 51-residue loop (IR3, 2487-2538) that functions as a molecular wedge between AAA3 and AAA4

• A 40-residue insertion in AAA5L (IR5, 3063-3103) forming a complex structure

• A C3HC4-type RING finger domain involved in protein interactions

This unique architectural organization makes RNF213 distinct from other E3 ligases and contributes to its specialized functions.

What applications can the biotin-conjugated RNF213 antibody be used for?

The biotin-conjugated RNF213 antibody is specifically designed for several research applications:

The antibody has been validated for detecting RNF213 in human samples, including serum, plasma, cell culture supernatants, and tissue sections . When using this antibody, it's important to optimize dilutions for each specific experimental system to obtain optimal results .

How should I design experiments to validate the specificity of RNF213 antibody binding?

To validate the specificity of the biotin-conjugated RNF213 antibody, a multi-step approach is recommended:

• Positive Control Selection: Use HUVEC cells as positive controls for Western blot and immunoprecipitation experiments, as these have been validated with RNF213 antibodies .

• Tissue Controls: For immunohistochemistry applications, human lymphoma tissue and human spleen tissue have been confirmed as appropriate positive controls .

• Antigen Competition Assay: Pre-incubate the antibody with the recombinant RNF213 antigen (amino acids 202-350) used for immunization before applying to your sample. Loss of signal indicates specificity.

• Knockdown/Knockout Validation: Perform experiments with RNF213 siRNA knockdown or CRISPR-Cas9 knockout cells and compare signal intensity with wild-type cells.

• Multiple Antibody Comparison: Validate results using alternative antibodies targeting different epitopes of RNF213.

This comprehensive validation approach ensures that signals detected in your experiments are genuinely attributable to RNF213.

What are the critical considerations for optimizing RNF213 detection in immunohistochemistry?

When performing immunohistochemistry with biotin-conjugated RNF213 antibody, consider these optimization steps:

• Antigen Retrieval: Use TE buffer at pH 9.0 for optimal epitope exposure. If results are unsatisfactory, citrate buffer at pH 6.0 can be used as an alternative .

• Antibody Dilution Range: Start with a dilution range of 1:20-1:200, then narrow to identify optimal concentration. The required dilution may vary with different tissue types .

• Blocking Endogenous Biotin: Since the antibody is biotin-conjugated, it's essential to block endogenous biotin using a biotin-blocking kit to prevent false-positive signals, particularly in biotin-rich tissues like liver and kidney.

• Detection System Selection: When using biotin-conjugated primary antibodies, employ streptavidin-HRP or streptavidin-fluorophore systems for visualization rather than secondary antibody approaches.

• Counterstain Compatibility: Select counterstains that don't interfere with the detection system. Hematoxylin works well for brightfield microscopy with DAB detection.

• Controls: Include both positive controls (human lymphoma or spleen tissue) and negative controls (primary antibody omission or isotype controls) to validate staining specificity .

How can I use the biotin-conjugated RNF213 antibody to investigate RNF213's role in ubiquitination pathways?

Investigating RNF213's ubiquitination activity requires specialized experimental approaches combining the biotin-conjugated antibody with other techniques:

• Co-Immunoprecipitation of Ubiquitination Complexes: Use the biotin-conjugated RNF213 antibody to pull down RNF213 and associated proteins. Analyze the precipitated complexes for the presence of E2 conjugating enzymes, particularly UbcH7, which has been identified as efficient in enabling ubiquitin transfer with RNF213 .

• In Vitro Ubiquitination Assays: Establish assays containing purified RNF213 (immunoprecipitated using the antibody), E1 activating enzyme, ATP, UbcH7 or UbcH5c as E2 enzymes, and ubiquitin. Detect auto-ubiquitination of RNF213 or ubiquitination of potential substrates .

• Domain-Specific Analysis: Recent structural studies revealed that RNF213 employs a unique RING-independent ubiquitination mechanism . Design experiments to investigate the roles of E3-back, E3-shell, E3-core, and CTD domains in this process.

• Substrate Identification: Combine the antibody with proximity-based labeling techniques (BioID or APEX) to identify potential substrates of RNF213's E3 ligase activity in relevant cell types.

The experimental design should account for RNF213's unique ubiquitination mechanism, which appears to operate via transthiolation rather than the canonical RING-dependent activation of E2-Ub conjugates .

What strategies can be implemented to study RNF213's role in Moyamoya disease using the biotin-conjugated antibody?

To investigate RNF213's involvement in Moyamoya disease using the biotin-conjugated antibody:

• Mutation-Specific Studies: Design experiments comparing wild-type RNF213 with the R4810K founder mutation. The antibody (targeting amino acids 202-350) will detect both variants, allowing for comparative analyses of expression levels, localization, and function .

• Vascular Cell Models: Employ the antibody in immunocytochemistry and biochemical assays using relevant cell types:

  • Human brain endothelial cells

  • Vascular smooth muscle cells

  • Pericytes isolated from patients with RNF213 mutations

• Signaling Pathway Analysis: Recent work has implicated RNF213 in hypoxia responses, lipid metabolism, NF-κB signaling, and angiogenesis . Use the antibody to track RNF213 expression and localization under different stimuli related to these pathways.

• Patient Sample Analysis: Apply the antibody in ELISA assays to quantify RNF213 levels in serum or plasma samples from Moyamoya disease patients compared to controls . This approach may reveal whether circulating RNF213 levels correlate with disease progression.

• Tissue Microarray Analysis: Perform immunohistochemistry on tissue microarrays containing samples from Moyamoya patients to evaluate RNF213 expression patterns in affected tissues.

This integrated approach will provide insights into how RNF213 mutations contribute to the pathophysiology of Moyamoya disease.

What are the common technical issues when using biotin-conjugated RNF213 antibody and how can they be resolved?

When using the biotin-conjugated RNF213 antibody, store at -20°C with aliquoting being unnecessary. The antibody remains stable for one year after shipment when stored properly .

How can I quantitatively analyze RNF213 expression levels using ELISA with the biotin-conjugated antibody?

For quantitative analysis of RNF213 using ELISA:

• Standard Curve Preparation: Prepare a standard curve using recombinant human RNF213 protein with concentrations ranging from 3.125 to 200 pg/ml as recommended for the Human RNF213 ELISA Kit .

• Sample Preparation:

  • For serum/plasma: Dilute samples appropriately based on expected concentration

  • For cell lysates: Use a standardized protein extraction method with protease inhibitors

  • For cell culture supernatants: Concentrate if necessary to ensure detection

• Assay Protocol:

  • Use the biotin-conjugated RNF213 antibody at a dilution of 1:500-1:1000

  • Include appropriate controls: blank, negative control, and positive control

  • Perform the assay in triplicate to ensure statistical significance

• Data Analysis:

  • Calculate protein concentration using the standard curve

  • For comparative studies, normalize data to total protein content

  • Use appropriate statistical methods to analyze differences between groups

• Assay Performance Metrics:

  • Sensitivity: 1.875 pg/ml

  • Detection range: 3.125-200 pg/ml

  • Intra-assay CV: <8%

  • Inter-assay CV: <10%

This approach provides a reliable method for quantifying RNF213 expression levels across different experimental conditions or clinical samples.

How can the biotin-conjugated RNF213 antibody be used to investigate the ATPase functionality of RNF213?

RNF213 uniquely combines E3 ubiquitin ligase activity with AAA ATPase functionality in a single polypeptide . To investigate this ATPase activity:

• Co-localization Studies: Use the biotin-conjugated RNF213 antibody in combination with fluorescently-labeled nucleotide analogs to visualize ATP binding sites.

• Activity Assays Following Immunoprecipitation:

  • Immunoprecipitate RNF213 using the biotin-conjugated antibody and streptavidin beads

  • Subject the immunoprecipitated protein to ATPase activity assays

  • Compare wild-type RNF213 with mutants affecting the AAA3 and AAA4 domains, which are catalytically competent

• Domain-Specific Analysis: Design experiments targeting the specific AAA domains identified in structural studies:

  • AAA2: Contains a nucleotide binding site that lacks catalytic residues but serves as molecular glue between AAA2 and AAA3

  • AAA3 and AAA4: The catalytically competent domains with all functional motifs

  • IR3 (2487-2538): The molecular wedge separating AAA3 and AAA4

• Conformational Change Studies: Investigate ATP-dependent conformational changes in RNF213 using limited proteolysis followed by immunoblotting with the biotin-conjugated antibody.

These approaches will help elucidate the relationship between RNF213's ATPase activity and its E3 ligase function, providing insights into its unique mechanism of action.

What are the best practices for studying interactions between RNF213 and the non-canonical Wnt signaling pathway?

The biotin-conjugated RNF213 antibody can be instrumental in investigating RNF213's role in the non-canonical Wnt signaling pathway in vascular development. Research shows that RNF213 mediates ubiquitination and degradation of FLNA and NFATC2 downstream of RSPO3, inhibiting the non-canonical Wnt pathway and promoting vessel regression . To study these interactions:

• Co-immunoprecipitation Experiments:

  • Use the biotin-conjugated antibody to pull down RNF213 and analyze co-precipitated proteins

  • Focus on detecting known interaction partners: FLNA, NFATC2, and components of the RSPO3 signaling pathway

  • Compare results between normal and Moyamoya disease-associated mutants

• Proximity Ligation Assays (PLA):

  • Combine the biotin-conjugated RNF213 antibody with antibodies against Wnt pathway components

  • Visualize protein-protein interactions in situ with subcellular resolution

  • Quantify interaction differences under various conditions (e.g., RSPO3 stimulation)

• Ubiquitination Analysis:

  • Immunoprecipitate FLNA or NFATC2 and probe for ubiquitination

  • Compare ubiquitination levels after RNF213 knockdown or overexpression

  • Examine how mutations in RNF213 affect substrate ubiquitination

• Functional Assays:

  • Assess vascular development in cell culture models while manipulating RNF213 levels

  • Quantify non-canonical Wnt pathway activity using reporter assays

  • Correlate RNF213 expression/localization with vessel regression markers

These approaches will provide mechanistic insights into how RNF213 regulates the non-canonical Wnt signaling pathway and vascular development, potentially revealing therapeutic targets for Moyamoya disease.