RNF213 Antibody, FITC conjugated

What is RNF213 and why is it significant in research?

RNF213 is an E3 ubiquitin-protein ligase involved in critical biological processes including angiogenesis and inflammatory signaling. The protein is particularly significant as it mediates ubiquitination and degradation of FLNA and NFATC2 downstream of RSPO3, thereby inhibiting the non-canonical Wnt signaling pathway and promoting vessel regression. Additionally, RNF213 possesses ATPase activity and plays roles in lipid metabolism and cell-autonomous immunity . The gene has gained prominence as a susceptibility gene for Moyamoya disease, making it an important target for vascular pathology research .

What are the primary applications for RNF213 antibody, FITC conjugated?

The FITC-conjugated RNF213 polyclonal antibody is primarily used in ELISA applications with recommended dilutions ranging from 1:100 to 1:500 . The fluorescent conjugation makes this antibody particularly valuable for applications requiring direct visualization without secondary antibody detection systems. While the primary validated application is ELISA, researchers may explore its utility in other fluorescence-based techniques such as immunofluorescence microscopy and flow cytometry, though additional validation would be necessary.

What are the key specifications of commercially available RNF213 antibody, FITC conjugated?

The commercially available RNF213 polyclonal antibody, FITC conjugated, is derived from rabbit hosts and demonstrates reactivity to human RNF213. It is an IgG isotype antibody that has been purified using Protein G purification methods. The immunogen used for producing this antibody is a recombinant human E3 ubiquitin-protein ligase RNF213 protein fragment (amino acids 202-350) . The antibody recognizes the protein also known by synonyms such as ALK lymphoma oligomerization partner on chromosome 17, with UniProt ID Q63HN8 .

How should I design experiments to detect RNF213 upregulation in response to inflammatory stimuli?

When designing experiments to detect RNF213 upregulation in response to inflammatory stimuli, consider the following methodology:

• Cell model selection: Human Umbilical Vein Endothelial Cells (HUVECs) or Human Coronary Artery Endothelial Cells (HCAECs) are validated models that show robust RNF213 responses to inflammatory stimuli .

• Stimulation protocol:

  • Treat cells with interferons (IFNA or IFNG) as primary inducers

  • For enhanced response, use combination treatment with TNFA and IFNG, which shows synergistic effects on RNF213 expression

  • Include appropriate time points (6-24 hours) based on the observed rapid elevation and decline in expression patterns

• Detection methods:

  • qRT-PCR for mRNA expression analysis

  • Western blot using the RNF213 antibody for protein level verification

  • Flow cytometry or immunofluorescence microscopy using FITC-conjugated antibody for cellular localization

• Controls:

  • Include unstimulated cells as negative controls

  • Use pathway inhibitors like LY294002 (PI3K-AKT inhibitor) or C16 (PKR inhibitor) to validate signaling mechanisms

What are the critical parameters for optimizing RNF213 antibody, FITC conjugated in ELISA assays?

When optimizing ELISA assays with FITC-conjugated RNF213 antibody, researchers should consider these critical parameters:

• Antibody dilution optimization:

  • Start with the recommended range of 1:100-1:500

  • Perform titration experiments to determine optimal signal-to-noise ratio

  • Consider the antigen concentration when determining optimal antibody dilution

• Blocking optimization:

  • Use protein-free blocking buffers to minimize background fluorescence

  • Extend blocking time (2-3 hours) if background signal is problematic

• Incubation conditions:

  • Protect from light during all steps involving the FITC-conjugated antibody

  • Maintain consistent temperature (typically room temperature or 4°C)

  • Optimize incubation time based on signal development

• Fluorescence detection:

  • Use appropriate excitation (approximately 495 nm) and emission (approximately 520 nm) filters

  • Include calibration curves with known standards

  • Consider photobleaching effects and minimize exposure to light

How can I validate the specificity of the RNF213 antibody, FITC conjugated in my experimental system?

To validate the specificity of FITC-conjugated RNF213 antibody in your experimental system:

• Positive controls:

  • Use cell lines with confirmed RNF213 expression (HUVECs are documented to express RNF213)

  • Include IFN-stimulated cells to enhance RNF213 expression as a positive control

• Negative controls:

  • Implement RNF213 knockdown using siRNA in your experimental system

  • Use isotype-matched FITC-conjugated control antibodies

  • Apply blocking peptides corresponding to the immunogen (202-350AA) to confirm specificity

• Cross-validation:

  • Compare results with alternative antibodies targeting different epitopes of RNF213

  • Verify results using orthogonal techniques (e.g., Western blot following immunoprecipitation)

  • Assess consistency with mRNA expression data

How can RNF213 antibody, FITC conjugated be utilized to investigate the role of RNF213 in Moyamoya disease pathogenesis?

For investigating RNF213's role in Moyamoya disease (MMD) pathogenesis using FITC-conjugated antibodies:

• Patient-derived samples:

  • Analyze fibroblasts or induced pluripotent stem cells (iPSCs) from MMD patients carrying the high-risk allele of RNF213 (c.14756G>A)

  • Compare RNF213 expression, localization, and response to inflammatory stimuli between patient and control samples

• Functional analysis:

  • Develop co-localization studies with vascular markers in tissue sections

  • Assess differences in RNF213 protein abundance, cellular distribution, or post-translational modifications

  • Implement flow cytometry with FITC-conjugated RNF213 antibody to quantify expression levels in different cell populations

• Inflammatory response models:

  • Create an inflammatory stimulation model using IFNG and TNFA to recapitulate potential disease triggers

  • Compare wild-type and MMD-associated RNF213 variant responses to these stimuli

  • Assess downstream effects on angiogenesis pathways and cell cycle progression

• Vessel formation assays:

  • Use the antibody to monitor RNF213 expression during in vitro angiogenesis assays

  • Correlate RNF213 levels with vessel formation capacity in MMD patient-derived cells

What methods can be used to study RNF213 protein interactions using FITC-conjugated antibodies?

To study RNF213 protein interactions using FITC-conjugated antibodies:

• Proximity ligation assays (PLA):

  • Combine FITC-conjugated RNF213 antibody with antibodies against potential interacting partners

  • Use PLA to visualize and quantify protein-protein interactions in situ

  • Focus on partners in the non-canonical Wnt signaling pathway like FLNA and NFATC2

• Co-immunoprecipitation with fluorescence detection:

  • Use conventional antibodies for immunoprecipitation of RNF213

  • Detect co-precipitated proteins using FITC-conjugated RNF213 antibody in subsequent analyses

  • Employ stringent washing conditions to minimize non-specific interactions

• FRET-based interaction studies:

  • Pair FITC-conjugated RNF213 antibody (donor) with antibodies against potential interaction partners conjugated with appropriate acceptor fluorophores

  • Analyze energy transfer to identify proteins in close proximity to RNF213

  • Validate interactions in different cellular compartments

• Dynamic interaction studies:

  • Implement live-cell imaging to track RNF213 interactions following inflammatory stimulation

  • Monitor temporal changes in protein associations during angiogenesis or in response to pathway modulators

How can researchers investigate the role of RNF213 in cell cycle regulation and angiogenesis?

To investigate RNF213's role in cell cycle regulation and angiogenesis:

• Cell cycle analysis protocol:

  • Implement flow cytometry with BrdU and 7-AAD labeling for cell cycle phase distribution

  • Compare RNF213 knockdown/overexpression effects on S-phase entry

  • Correlate RNF213 expression with cyclins (CCNA2, CCNB1, CCNE1) using co-staining approaches

• Angiogenesis models:

  • Tube formation assays with endothelial cells expressing different levels of RNF213

  • Spheroid-based sprouting assays to assess the impact on vessel formation

  • In vivo models using zebrafish or mouse models with modulated RNF213 expression

• Mechanistic studies:

  • Investigate ubiquitination targets of RNF213 using immunoprecipitation followed by mass spectrometry

  • Assess the impact of AKT and PKR pathway inhibitors on RNF213-mediated angiogenesis

  • Study the effects of inflammatory stimuli (IFNG, TNFA) on RNF213 expression during vessel formation

• Single-cell analysis:

  • Employ FITC-conjugated RNF213 antibody in flow cytometry to correlate RNF213 expression with cell cycle phases

  • Analyze heterogeneity in RNF213 expression within endothelial cell populations during angiogenesis

What are common problems and solutions when using RNF213 antibody, FITC conjugated?

How should RNF213 antibody, FITC conjugated be stored and handled to maintain optimal activity?

For optimal maintenance of FITC-conjugated RNF213 antibody activity:

• Storage conditions:

  • Store at -20°C as recommended for antibody products

  • Maintain in darkness to prevent photobleaching of the FITC conjugate

  • Ensure storage buffer contains 0.02% sodium azide and 50% glycerol (pH 7.3) for stability

  • Antibody will remain stable for one year after shipment when properly stored

• Handling practices:

  • Avoid repeated freeze-thaw cycles that can degrade antibody activity

  • Aliquot upon first thaw to minimize freeze-thaw events

  • Protect from light during all handling steps

  • Allow to equilibrate to room temperature before opening to avoid condensation

• Working solution preparation:

  • Dilute only the amount needed for immediate use

  • Use high-quality diluents free of contaminants

  • Prepare fresh working solutions for each experiment

  • Return stock solution to -20°C storage promptly after use

How can researchers address the technical challenges of detecting the high molecular weight RNF213 protein (374 kDa)?

Addressing the technical challenges of detecting high molecular weight RNF213 protein (374 kDa):

• Protein extraction optimization:

  • Use specialized lysis buffers containing protease inhibitors to prevent degradation

  • Employ gentle mechanical disruption techniques to preserve protein integrity

  • Consider native extraction methods to maintain protein conformation

• Gel electrophoresis adaptations:

  • Utilize low percentage (3-6%) polyacrylamide gels or gradient gels

  • Extend running time at lower voltage to allow proper separation

  • Consider specialized large-protein electrophoresis systems

  • Use high molecular weight markers that extend beyond 250 kDa

• Transfer modifications for Western blotting:

  • Implement extended transfer times or semi-dry transfer systems

  • Reduce methanol concentration in transfer buffer to improve large protein transfer

  • Consider specialized transfer conditions designed for high molecular weight proteins

• Detection strategy:

  • Employ signal amplification methods to enhance detection sensitivity

  • Utilize longer primary antibody incubation times (overnight at 4°C)

  • Consider direct detection using the FITC-conjugated antibody to eliminate secondary antibody limitations

How should researchers interpret variations in RNF213 expression patterns across different experimental conditions?

When interpreting variations in RNF213 expression patterns:

• Baseline expression analysis:

  • Recognize tissue-specific expression patterns (highest in heart tissue based on mouse models)

  • Consider cell type-specific variations in basal expression levels

  • Normalize to appropriate housekeeping genes when comparing across tissues or cell types

• Stimulus-induced expression changes:

  • Interpret time-course data carefully; RNF213 expression peaks around 6 hours post-stimulation with IFNG and TNFA and rapidly declines within 24 hours

  • Consider synergistic effects when multiple cytokines are present (TNFA+IFNG > IFNG alone)

  • Analyze pathway-specific effects using inhibitor studies (PI3K-AKT and PKR pathways)

• Genetic variant considerations:

  • Note that MMD-associated variants (c.14756G>A) may not affect basal expression or cytokine responsiveness at the mRNA level

  • Investigate potential differences in protein function, localization, or stability rather than expression levels

• Correlation with functional outcomes:

  • Connect RNF213 expression changes with functional endpoints such as cell cycle progression, angiogenesis, or inflammatory response

  • Analyze relationships between RNF213 levels and expression of cyclins (CCNA2, CCNB1, CCNE1)

What controls are essential when conducting research with RNF213 antibody, FITC conjugated?

Essential controls for research with FITC-conjugated RNF213 antibody include:

• Antibody specificity controls:

  • Isotype-matched FITC-conjugated control antibodies to assess non-specific binding

  • RNF213 knockdown samples to confirm signal specificity

  • Immunogen blocking experiments using recombinant RNF213 protein (202-350AA)

• Fluorescence controls:

  • Unstained samples to establish autofluorescence baseline

  • Single-color controls for compensation in multicolor experiments

  • Fluorescence minus one (FMO) controls when using multiple fluorescent antibodies

• Biological controls:

  • Unstimulated versus IFNG/TNFA-stimulated endothelial cells to demonstrate inducible expression

  • Pathway inhibitor treatments (LY294002, C16) to validate signaling mechanisms

  • Comparison between tissues with known differential expression (heart versus other tissues)

• Technical controls:

  • Serial dilution of antibody to establish optimal working concentration

  • Positive control samples (HUVECs) with established RNF213 expression

  • Cross-validation with alternative detection methods (qRT-PCR, non-conjugated antibodies)

How can researchers ensure reproducibility in studies involving RNF213 antibody, FITC conjugated?

To ensure reproducibility in studies with FITC-conjugated RNF213 antibody:

• Antibody validation and documentation:

  • Maintain detailed records of antibody lot numbers and validation experiments

  • Establish consistent performance criteria across batches

  • Implement standardized protocols for antibody handling and storage

• Experimental standardization:

  • Standardize cell culture conditions including passage number, confluence, and serum batch

  • Establish consistent stimulation protocols for IFNG and TNFA treatments

  • Document instrument settings for fluorescence detection (exposure time, gain, filters)

• Quantification methods:

  • Implement automated image analysis algorithms to reduce subjective interpretation

  • Use consistent gating strategies for flow cytometry experiments

  • Apply appropriate statistical methods for data analysis with sufficient biological replicates

• Protocol transparency:

  • Document detailed methods including antibody dilutions, incubation times, and buffer compositions

  • Share raw data and analysis pipelines when publishing

  • Precisely report experimental conditions that may affect RNF213 expression (cytokine stimulation, pathway inhibitors)