RANBP9 Antibody, FITC conjugated

What is RANBP9 and what cellular functions does it regulate?

RANBP9 functions primarily as a scaffolding protein and adapter molecule coupling membrane receptors to intracellular signaling pathways. It plays critical roles in multiple cellular processes including:

• Mediation of cell spreading and actin cytoskeleton rearrangement

• Core component of the CTLH E3 ubiquitin-protein ligase complex that selectively accepts ubiquitin from UBE2H

• Regulation of transcription factor HBP1 through ubiquitination and proteasomal degradation

• Integration of signaling between β-integrins, LRP, and amyloid precursor protein (APP)

Subcellularly, RANBP9 localizes to the cytoplasm, nucleus, and cell membrane, consistent with its diverse functions in cellular signaling pathways .

What are the recommended applications for RANBP9 FITC-conjugated antibodies?

RANBP9 FITC-conjugated antibodies are primarily optimized for the following applications:

For immunofluorescence applications utilizing the FITC conjugate, the antibody enables direct detection without secondary antibodies, streamlining experimental workflows and reducing background in multi-color imaging protocols .

How should RANBP9 antibodies be stored to maintain optimal activity?

To maintain optimal reactivity and fluorescence signal of RANBP9 FITC-conjugated antibodies:

• Store at -20°C in the recommended storage buffer (typically PBS with 0.01-0.02% sodium azide, 1% BSA, and 50% glycerol at pH 7.3-7.4)

• Divide into small aliquots to avoid repeated freeze-thaw cycles which significantly degrade antibody performance

• When thawed for use, keep on ice and protected from light to preserve FITC fluorescence

• Most preparations remain stable for approximately one year after shipment when properly stored

Monitoring fluorescence intensity in positive controls before experimental use is recommended, particularly for antibodies stored longer than 6 months.

What are the recommended protocols for immunofluorescence detection of RANBP9?

For optimal immunofluorescence detection using FITC-conjugated RANBP9 antibodies:

• Fix cells using 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

• Block with 5% normal serum in PBS for 1 hour at room temperature

• Apply FITC-conjugated RANBP9 antibody at dilutions of 1:50-1:500 in blocking buffer

• Incubate overnight at 4°C or for 1-2 hours at room temperature in a humidified chamber

• Wash extensively with PBS (3-5 times for 5 minutes each)

• Counterstain nuclei with DAPI if desired

• Mount with anti-fade mounting medium

When imaging, use appropriate filter sets for FITC (excitation ~495 nm, emission ~520 nm) and minimize exposure to prevent photobleaching. Visualization should reveal RANBP9 distribution in cytoplasmic, nuclear, and membrane compartments, with potential enrichment in focal adhesion sites when co-stained with adhesion markers .

How can researchers design experiments to study RANBP9's role in integrin-mediated adhesion?

To study RANBP9's effects on integrin-mediated adhesion, consider the following experimental approach:

• Comparative cell adhesion assays:

  • Plate control and RANBP9-overexpressing or knockdown cells on integrin substrates (fibronectin, collagen, etc.)

  • Quantify adhesion after 15-60 minutes by washing, fixing, and counting attached cells

  • Compare spreading area using phase-contrast microscopy or fluorescent cytoskeletal markers

• Focal adhesion analysis:

  • Co-stain for RANBP9 and focal adhesion proteins (paxillin, vinculin, talin)

  • Quantify focal adhesion size, number, and distribution using image analysis software

  • Monitor focal adhesion turnover using live-cell imaging of fluorescently tagged adhesion proteins

• Surface biotinylation assays:

  • Label surface proteins with cleavable biotin

  • Immunoprecipitate with antibiotin antibody

  • Analyze β1-integrin and LRP cell surface levels by western blotting

  • Perform endocytosis assays (with a 3-minute internalization period) to measure accelerated endocytosis

This approach has revealed that RANBP9 overexpression dramatically disrupts integrin-dependent cell attachment and spreading while decreasing Pyk2/paxillin signaling and focal adhesion assembly. Conversely, RANBP9 knockdown promotes these processes, suggesting a role in regulating the endocytosis of key adhesion receptors .

How does RANBP9 expression influence DNA damage response pathways in cancer cells?

RANBP9 has emerged as a critical modulator of DNA damage response (DDR) pathways, particularly in non-small cell lung cancer (NSCLC). Researchers investigating this relationship should consider:

• RANBP9 exists as both a target and enabler of ataxia telangiectasia mutated (ATM) kinase signaling

• RANBP9 depletion abates ATM activation and downstream targets including p53 signaling

• RANBP9 knockout cells exhibit selective sensitivity patterns:

  • Increased sensitivity to ataxia and telangiectasia-related (ATR) kinase inhibition

  • Minimal response to ATM inhibition

  • Enhanced sensitivity to Poly(ADP-ribose)-Polymerase (PARP) inhibitors, resulting in a "BRCAness-like" phenotype

These findings suggest RANBP9 expression levels may predict patient response to specific DNA damaging agents. Methodologically, researchers can manipulate RANBP9 expression through knockout, knockdown, or overexpression systems to assess sensitivity to various DDR-targeting therapeutic agents and analyze downstream signaling pathways through phosphorylation-specific antibodies and functional assays .

What are the methodological approaches to study RANBP9's scaffolding functions in different cellular compartments?

RANBP9's diverse cellular functions stem from its ability to scaffold multiple protein complexes across different cellular compartments. To investigate these functions:

• Subcellular fractionation approaches:

  • Isolate cytoplasmic, nuclear, and membrane fractions using differential centrifugation

  • Analyze RANBP9 distribution and interaction partners in each fraction

  • Perform co-immunoprecipitation with FITC-conjugated RANBP9 antibodies followed by mass spectrometry

• Proximity labeling techniques:

  • Generate RANBP9 fusion constructs with BioID or APEX2

  • Identify proteins in close proximity to RANBP9 in living cells

  • Compare interactome differences across cellular compartments

• Live-cell imaging strategies:

  • Create fluorescent protein-tagged RANBP9 constructs

  • Perform FRAP (Fluorescence Recovery After Photobleaching) to assess mobility

  • Conduct FRET analyses with potential binding partners

  • Track dynamic redistribution following various cellular stimuli

These approaches help elucidate how RANBP9 coordinates between its roles at receptor tyrosine kinases at the membrane, intracellular messengers in the cytoplasm, and transcription factors in the nucleus – all crucial contexts for its multiple biological functions .

What are common issues with immunofluorescence detection of RANBP9 and how can they be addressed?

When using FITC-conjugated RANBP9 antibodies for immunofluorescence, researchers may encounter several technical challenges:

For optimal antigen retrieval when performing immunohistochemistry on formalin-fixed tissues, TE buffer at pH 9.0 is recommended, though citrate buffer at pH 6.0 can serve as an alternative .

How should researchers validate RANBP9 antibody specificity in experimental systems?

Thorough validation of RANBP9 antibody specificity is critical for experimental reliability. A comprehensive validation approach includes:

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • Run parallel assays with blocked and unblocked antibody

  • Expect significant signal reduction in blocked samples

• Genetic knockdown/knockout controls:

  • Generate RANBP9 siRNA knockdown or CRISPR knockout cell lines

  • Compare antibody reactivity in wild-type versus knockdown/knockout cells

  • Expect substantially reduced signal in depleted samples

• Cross-platform verification:

  • Confirm findings across multiple detection methods (WB, IP, IF)

  • Verify observed molecular weight matches expected size (80-90 kDa observed versus calculated 78 kDa)

  • Analyze recognized isoforms across different tissue/cell types

• Multiple antibody comparison:

  • Test antibodies targeting different RANBP9 epitopes

  • Compare detection patterns and subcellular localization

  • Consistent results across antibodies increase confidence in specificity

When specifically using FITC-conjugated antibodies, include appropriate controls for autofluorescence and test unconjugated primary antibodies with secondary detection for comparison .

How can RANBP9 antibodies be applied to investigate its role in neurodegenerative processes?

RANBP9 has significant implications in neurodegenerative conditions, particularly Alzheimer's disease (AD). For researchers investigating these connections:

• Co-localization studies in neural tissue:

  • Utilize FITC-conjugated RANBP9 antibodies to examine co-localization with APP, BACE1, and LRP in primary neurons or brain sections

  • Analyze enrichment patterns around amyloid plaques in AD models

  • Quantify changes in cellular distribution during disease progression

• Neurite outgrowth and arborization analysis:

  • Culture primary hippocampal neurons from control and RANBP9-transgenic mice

  • Trace neurite development using FITC-conjugated RANBP9 alongside cytoskeletal markers

  • Measure neurite length, branching complexity, and spine morphology

  • Correlate changes with surface levels of β1-integrin, LRP, and APP

• Endocytosis dynamics in neurons:

  • Perform surface biotinylation assays on primary neurons

  • Track internalization rates of APP, LRP, and integrins

  • Correlate endocytosis rates with Aβ generation and neurite complexity

Previous studies have demonstrated that primary hippocampal neurons from RANBP9-transgenic mice exhibit severely reduced levels of surface β1-integrin, LRP, and APP, with corresponding reductions in neurite arborization, suggesting RANBP9's role in linking endocytic regulation to neurodegeneration .

What methodological considerations are important when studying RANBP9 as a potential therapeutic target in cancer?

When investigating RANBP9 as a therapeutic target in cancer, particularly non-small cell lung cancer (NSCLC), researchers should consider:

• Expression profiling across cancer stages:

  • Perform immunohistochemistry on tissue microarrays using optimized RANBP9 antibodies (1:20-1:200 dilution)

  • Correlate expression patterns with clinical outcomes and treatment responses

  • Compare tumor tissues with matched adjacent normal tissues

• Functional modulation strategies:

  • Design targeted knockdown approaches (siRNA, shRNA)

  • Develop specific inhibitors of RANBP9-protein interactions

  • Create dominant negative constructs targeting critical domains

• Combination therapy assessment:

  • Test RANBP9 inhibition in combination with standard chemotherapeutics

  • Evaluate synergy with DNA-damaging agents, particularly platinum compounds

  • Explore selective sensitization to ATR inhibitors and PARP inhibitors

• Biomarker development:

  • Investigate RANBP9 expression as a predictive biomarker for responsiveness to specific DNA damaging agents

  • Develop quantitative assays for RANBP9 levels in patient samples

  • Correlate expression with treatment outcomes in retrospective and prospective studies

How can researchers design experiments to investigate RANBP9's ubiquitin-proteasome related functions?

RANBP9 serves as a core component of the CTLH E3 ubiquitin-protein ligase complex that mediates ubiquitination and proteasomal degradation of specific target proteins. To study these functions:

• Proteasome inhibition studies:

  • Treat cells with proteasome inhibitors (MG132, bortezomib)

  • Analyze accumulation of RANBP9 and its binding partners

  • Perform co-immunoprecipitation with FITC-conjugated RANBP9 antibodies to identify stabilized complexes

• Ubiquitination assays:

  • Design in vitro and in vivo ubiquitination assays

  • Express tagged ubiquitin constructs

  • Precipitate ubiquitinated proteins and probe for specific targets

  • Analyze different ubiquitin chain topologies (K48, K63, etc.)

• CTLH complex analysis:

  • Investigate interactions between RANBP9 and other CTLH components

  • Perform sequential immunoprecipitation to isolate intact complexes

  • Analyze substrate selectivity and specificity

  • Map regulatory domains through truncation and mutation approaches

These approaches will help elucidate how RANBP9 contributes to protein homeostasis through selective protein degradation, particularly of transcription factors like HBP1, which may have significant implications for various cellular processes and disease states .

What considerations are important when designing multiplexed imaging experiments involving RANBP9 antibodies?

For researchers planning multiplexed imaging experiments to study RANBP9 in complex cellular contexts:

• Spectral compatibility planning:

  • When using FITC-conjugated RANBP9 antibodies (excitation ~495 nm, emission ~520 nm), select compatible fluorophores for co-staining

  • Avoid significant spectral overlap with FITC by choosing far-red (Cy5, Alexa 647) or far-blue (Pacific Blue) fluorophores for other targets

  • If needed, use linear unmixing algorithms to separate overlapping signals

• Sequential staining approaches:

  • For highly multiplexed imaging (>4 targets), consider sequential staining with bleaching or antibody stripping between rounds

  • Perform RANBP9 detection in early rounds to minimize signal degradation of the FITC conjugate

  • Document precise stage positions for accurate image registration between rounds

• Sample preparation optimizations:

  • Test fixation conditions that preserve both RANBP9 epitopes and co-target antigens

  • Optimize permeabilization to allow antibody access while maintaining structural integrity

  • Consider tissue clearing techniques for thick sections or 3D cultures

  • Implement appropriate blocking steps to minimize cross-reactivity

• Validation controls:

  • Include single-stain controls for each fluorophore

  • Prepare secondary-only controls to assess background

  • Include biological controls with known expression patterns

  • Implement computational approaches to correct for autofluorescence

These considerations ensure reliable detection of RANBP9 alongside its interaction partners or downstream effectors in complex biological specimens, enabling more comprehensive understanding of its multiple cellular functions .