RNF130 Antibody

What is RNF130 and why is it significant in molecular research?

RNF130 (Ring Finger Protein 130) is a 419-amino acid protein (46.4 kDa) that functions as an E3 ubiquitin ligase. It contains a RING finger motif and is ubiquitously expressed across various tissue types with subcellular localization in both membrane and cytoplasm . Its significance stems from its role in regulating LDLR (Low-Density Lipoprotein Receptor) availability through ubiquitination, directly affecting plasma LDL cholesterol levels . Recent studies have identified RNF130 as a novel post-translational regulator of cholesterol metabolism, making it a potential therapeutic target for lipid disorders . Additionally, RNF130 may regulate programmed cell death in hematopoietic cells and has been found upregulated in certain pathological conditions including hydatidiform mole tissue and esophageal adenocarcinoma .

What types of RNF130 antibodies are available for research applications?

RNF130 antibodies are predominantly polyclonal antibodies produced in rabbits, targeting various epitopes of the human RNF130 protein . The available formats include:

These antibodies target different regions of RNF130, including internal regions, C-terminal domains, and specific amino acid sequences (e.g., AA 44-93) . Western blotting is the most validated application, typically using dilutions of 0.04-0.4 μg/mL for optimal results .

How are RNF130 antibodies validated for research applications?

Validation of RNF130 antibodies follows a multi-step process to ensure specificity and reliability :

• Western blot analysis: Performed on panels of human tissues and cell lines to evaluate antibody specificity at the expected molecular weight of 46.4 kDa (±20%)

• Protein array testing: Antibodies are tested against arrays containing 384 different antigens including the target to analyze cross-reactivity

• Immunohistochemistry validation: Tested against 44 normal human tissues and 20 common cancer tissue types

• Enhanced validation methods may include:

  • Genetic validation through knockout/knockdown models

  • Recombinant expression validation

  • Independent antibody validation with multiple antibodies targeting different epitopes

  • Orthogonal validation comparing protein and RNA expression

  • Capture MS validation

For revalidation of antibodies with initial unreliable results, overexpression lysates are commonly employed .

What are the optimal conditions for using RNF130 antibodies in Western blotting?

For optimal Western blot detection of RNF130:

• Sample preparation:

  • Homogenize tissue samples in RIPA buffer containing protease inhibitor cocktail (Complete MINI EDTA-free tablets, calpain inhibitor, and PMSF)

  • Load 25-50 μg of protein per lane on SDS-PAGE gels

• Transfer conditions:

  • Transfer to PVDF membrane for optimal protein binding

  • Use standard transfer conditions (typically 100V for 1 hour or 30V overnight)

• Antibody dilutions:

  • Primary RNF130 antibody: 1:1000 dilution (0.04-0.4 μg/mL)

  • Secondary HRP-conjugated antibody: 1:10,000 dilution

• Detection system:

  • Enhanced chemiluminescence (ECL) is recommended

  • Expected band size: approximately 46.4 kDa

  • Note that glycosylated forms may appear at slightly higher molecular weights

• Controls:

  • Positive control: Liver tissue shows high expression

  • Negative control: RNF130 knockout/knockdown samples when available

How can RNF130 antibodies be used to study protein-protein interactions?

RNF130 antibodies are valuable tools for studying protein-protein interactions, particularly in the context of ubiquitination pathways:

• Co-immunoprecipitation (Co-IP) protocol:

  • Prepare cell lysates in RIPA buffer with protease inhibitors

  • Clear lysates by centrifugation (10,000 × g, 10 min, 4°C)

  • Quantify protein concentration using BCA assay

  • Incubate equal amounts of protein with anti-RNF130 antibody (1:1000) overnight at 4°C

  • Add protein-G agarose beads for an additional 2 hours

  • Wash beads 3× with RIPA buffer containing protease inhibitors

  • Elute bound proteins with Laemmli buffer at 70°C for 30 minutes

• Ubiquitination assays:

  • Transfect cells with HA-tagged ubiquitin and target protein (e.g., LDLR-GFP)

  • Co-express RNF130 or RNF130 mutants (e.g., C304A RING domain mutant)

  • Immunoprecipitate the target protein (e.g., with anti-GFP antibodies)

  • Detect ubiquitination with anti-HA antibodies

  • Use RNF130 antibodies to confirm expression levels in input samples

• Controls for specificity:

  • Include non-specific IgG controls

  • Use RING domain mutants (C304A) as negative controls for E3 ligase activity

  • Include lysates from RNF130 knockout/knockdown cells

What immunohistochemistry protocols work best with RNF130 antibodies?

For optimal immunohistochemistry results with RNF130 antibodies:

• Tissue preparation:

  • Formalin-fixed, paraffin-embedded sections (5 μm thickness)

  • Antigen retrieval: Citrate buffer (pH 6.0), heat-induced epitope retrieval recommended

• Staining protocol:

  • Deparaffinize and rehydrate sections through xylene and graded alcohols

  • Block endogenous peroxidase activity with 3% H₂O₂

  • Block non-specific binding with 5% normal serum from the same species as the secondary antibody

  • Primary antibody incubation: 1:200-1:500 dilution, overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-rabbit IgG, 30 minutes at room temperature

  • Visualization: DAB (3,3'-diaminobenzidine) substrate

  • Counterstain: Hematoxylin

• Positive control tissues:

  • Liver tissue shows strong expression

  • Leukocytes demonstrate consistent expression

• Expected staining pattern:

  • Membranous and cytoplasmic staining

  • Variable intensity across different cell types

How can RNF130 antibodies be used to study its role in LDLR regulation and cholesterol metabolism?

For investigating RNF130's role in LDLR regulation:

• LDLR ubiquitination analysis:

  • Perform in vitro ubiquitination assays using purified components:

    • Recombinant E1, E2, and RNF130 (E3)

    • Purified LDLR or LDLR domains

    • Ubiquitin and ATP

  • Detect ubiquitinated LDLR using anti-LDLR and anti-ubiquitin antibodies

  • Use RNF130 antibodies to confirm the presence of RNF130 in the reaction

• LDLR trafficking studies:

  • Perform immunofluorescence co-localization studies:

    • Co-stain cells with anti-LDLR and anti-RNF130 antibodies

    • Use markers for different cellular compartments (plasma membrane, endosomes, etc.)

  • Track LDLR internalization and recycling in the presence or absence of RNF130

  • Quantify cell surface LDLR using cell surface biotinylation assays and RNF130 antibodies to correlate with RNF130 expression levels

• In vivo studies:

  • Use RNF130 antibodies to verify RNF130 expression/knockdown in various models:

    • Antisense oligonucleotide (ASO) treatment

    • Germline deletion models

    • AAV-CRISPR-mediated disruption

  • Correlate RNF130 protein levels with hepatic LDLR abundance and plasma LDL-C levels

  • Examine tissue-specific effects using immunohistochemistry with RNF130 antibodies

What approaches are recommended for studying RNF130 in disease models?

For studying RNF130 in disease contexts:

• Cancer research applications:

  • Quantitative analysis in tissue microarrays:

    • RNF130 is upregulated in Barrett esophagus (BE) and esophageal adenocarcinoma (EAC)

    • Use RNF130 antibodies at 1:200-1:500 dilution for IHC

    • Quantify expression using H-scoring or digital image analysis

  • Correlation with disease progression:

    • Compare RNF130 expression between normal, premalignant, and malignant tissues

    • Correlate with clinical outcomes and other molecular markers

• Cardiovascular disease models:

  • Atherosclerosis models:

    • Use RNF130 antibodies to monitor expression in atherosclerotic plaques

    • Correlate with LDLR levels and plasma cholesterol

  • High-fat diet studies:

    • Monitor RNF130 expression changes in response to dietary challenges

    • Track correlation with lipid profiles and LDLR expression

• Experimental knockdown/overexpression validation:

  • For knockdown validation:

    • Western blotting with RNF130 antibodies (1:1000 dilution)

    • qPCR to correlate protein with mRNA levels

  • For overexpression studies:

    • Compare wildtype RNF130 with RING domain mutants (C304A)

    • Use FLAG-tagged constructs with both anti-FLAG and anti-RNF130 antibodies

How can researchers troubleshoot inconsistent RNF130 antibody staining results?

When encountering inconsistent RNF130 antibody staining:

• Epitope masking issues:

  • RNF130 undergoes glycosylation which may mask epitopes

  • Try multiple antibodies targeting different regions of the protein

  • For glycosylated proteins, consider enzymatic deglycosylation (PNGase F treatment) before analysis

• Fixation-dependent epitope accessibility:

  • Compare different fixation methods:

    • 4% paraformaldehyde (10-15 minutes)

    • Methanol fixation (-20°C, 10 minutes)

    • Acetone fixation (room temperature, 5 minutes)

  • Optimize antigen retrieval methods (citrate buffer vs. EDTA buffer)

• Validation across multiple platforms:

  • Confirm expression using orthogonal methods:

    • Western blot

    • qPCR

    • Mass spectrometry

  • Use genetic models (knockout/knockdown) as negative controls

  • Consider testing for specificity on protein arrays

• Sample-specific considerations:

  • Fresh vs. frozen vs. fixed tissues may require different antibody dilutions

  • Autofluorescence can be a problem in some tissues - use appropriate quenching methods

  • High endogenous peroxidase activity may require extended blocking steps

  • Consider tissue-specific expression levels when optimizing protocols

How should researchers quantify and normalize RNF130 expression in Western blot analyses?

For accurate quantification of RNF130 in Western blots:

• Loading control selection:

  • GAPDH or β-actin for general normalization

  • PDI (Protein Disulfide Isomerase) for ER-localized proteins

  • Na⁺/K⁺ ATPase for membrane proteins

  • Consider compartment-specific controls since RNF130 localizes to both membrane and cytoplasm

• Densitometry methods:

  • Use software like ImageJ, Image Lab, or AI600 Imager quantification tools

  • Ensure linear range of detection (avoid saturated signals)

  • Subtract background using local background correction

  • Normalize RNF130 band intensity to loading control

• Technical considerations:

  • Run a dilution series of a positive control to establish a standard curve

  • Include at least 3 biological replicates for statistical validity

  • For glycosylated RNF130, consider quantifying all bands within the 45-55 kDa range

  • Report both individual and combined band intensities when multiple bands are present

What are the best practices for analyzing RNF130 immunostaining in tissues?

For rigorous analysis of RNF130 immunostaining:

• Scoring systems:

  • Semi-quantitative H-score (combines intensity and percentage of positive cells)

  • Digital pathology quantification (for more objective assessment)

  • Consider both membrane and cytoplasmic staining separately

• Comparison across tissues:

  • RNF130 is ubiquitously expressed but with variable intensity

  • Liver and leukocytes show high expression and can serve as internal positive controls

  • Use standardized tissue microarrays for comparison across multiple samples

• Co-localization analysis:

  • For fluorescence microscopy, use Pearson's or Mander's coefficients

  • Compare RNF130 localization with organelle markers:

    • Plasma membrane (Na⁺/K⁺ ATPase)

    • Endoplasmic reticulum (Calnexin, PDI)

    • Golgi (GM130)

    • Endosomes (EEA1, Rab5, Rab7, Rab11)

• Documentation guidelines:

  • Include full tissue section images at low magnification

  • Provide higher magnification of representative areas

  • Document all microscope settings, exposure times, and post-processing details

How can researchers validate the specificity of their RNF130 antibody-based findings?

To ensure the validity of RNF130 antibody-based research:

• Multiple antibody approach:

  • Use at least two independent antibodies targeting different epitopes

  • Compare antibodies from different vendors or different host species

  • Confirm similar patterns across different detection methods

• Genetic validation:

  • CRISPR/Cas9 knockout or knockdown validation

  • siRNA or shRNA knockdown

  • Rescue experiments with ectopic expression of RNF130

  • Compare wildtype and RING domain mutants (C304A) to validate functional observations

• Cross-species validation:

  • RNF130 is conserved across species (human, mouse, rat, bovine, etc.)

  • Compare expression patterns in orthologous tissues from different species

  • Be aware of potential species-specific differences in glycosylation or other post-translational modifications

• Technical controls:

  • Peptide competition assays to confirm specificity

  • Pre-adsorption of antibody with recombinant antigen

  • Secondary-only controls to rule out non-specific binding

  • Isotype controls to confirm specificity of binding

How can RNF130 antibodies be utilized in high-throughput screening approaches?

For high-throughput applications involving RNF130:

• Automated immunofluorescence platforms:

  • Cell-based screening of compounds affecting RNF130 expression or localization

  • Use fluorophore-conjugated anti-RNF130 antibodies (488, 555, 647) for direct detection

  • Multiplex with anti-LDLR and other markers to study pathway interactions

  • Quantify using high-content imaging systems

• ELISA-based screening:

  • Develop sandwich ELISA using capture and detection anti-RNF130 antibodies

  • Screen for compounds modulating RNF130 protein levels

  • Develop phospho-specific antibodies if regulatory phosphorylation sites are identified

• Protein array applications:

  • Use purified RNF130 to identify novel binding partners

  • Employ anti-RNF130 antibodies to detect RNF130 in tissue or serum samples across large cohorts

  • Screen for autoantibodies against RNF130 in patient samples

What are the latest methodologies for studying RNF130's E3 ligase activity?

Advanced methods for investigating RNF130's E3 ligase function:

• In vitro reconstituted ubiquitination systems:

  • Use purified components (E1, E2, RNF130, substrates like LDLR)

  • Monitor ubiquitin chain formation using anti-ubiquitin antibodies

  • Analyze chain topology (K48 vs. K63 linkages) using linkage-specific antibodies

  • Detect RNF130 auto-ubiquitination using anti-RNF130 antibodies

• Proximity-based labeling approaches:

  • BioID or TurboID fusion with RNF130 to identify proximal proteins in living cells

  • APEX2 fusion for temporal control of proximity labeling

  • Use anti-RNF130 antibodies to verify expression of fusion proteins

• Proteomic identification of substrates:

  • Stable isotope labeling with amino acids in cell culture (SILAC)

  • Tandem ubiquitin binding entities (TUBEs) to enrich ubiquitinated proteins

  • Compare ubiquitinomes in RNF130-overexpressing vs. RNF130-knockout models

  • Confirm findings using RNF130 antibodies in validation experiments

• Fluorescence-based real-time assays:

  • FRET-based ubiquitination sensors

  • Live-cell imaging of fluorescently tagged RNF130 and substrates

  • Correlate with fixed-cell immunostaining using RNF130 antibodies

How might RNF130 antibodies contribute to potential therapeutic developments?

Potential therapeutic applications involving RNF130 antibodies:

• Target validation:

  • Use RNF130 antibodies to confirm target engagement by small molecules

  • Monitor RNF130 expression changes in response to therapeutic interventions

  • Correlate RNF130 levels with LDLR expression and plasma LDL-C in clinical samples

• Biomarker development:

  • Evaluate RNF130 as a potential biomarker for:

    • Lipid disorders

    • Esophageal adenocarcinoma progression

    • Other conditions where RNF130 is dysregulated

  • Develop standardized immunoassays suitable for clinical samples

• Therapeutic antibody development pipeline:

  • Use research-grade antibodies to identify accessible epitopes

  • Develop neutralizing antibodies targeting the RING domain

  • Engineer antibody-drug conjugates targeting RNF130-expressing cells in disease contexts

  • Intrabodies targeting intracellular RNF130 (delivered via gene therapy approaches)

• Monitoring therapeutic efficacy:

  • Track changes in RNF130 and LDLR expression during lipid-lowering therapy

  • Correlate with clinical outcomes in cardiovascular disease studies

  • Use in conjunction with plasma LDL-C measurements to assess treatment response