RANBP10 Antibody, HRP conjugated

What is RANBP10 and why is it important for research?

RANBP10 (RAN binding protein 10) functions as a guanine nucleotide exchange factor (GEF) for RAN GTPase, playing essential roles in hemostasis and microtubule dynamics, particularly in platelets. It serves as an adapter protein that couples membrane receptors to intracellular signaling pathways and enhances transactivation activity of specific nuclear receptors. RANBP10 is part of the CTLH (C-Terminal to LisH) complex, an evolutionarily conserved multi-subunit E3 ligase complex that regulates various cellular functions . Recent research has indicated RANBP10 overexpression in certain cancers, including glioblastoma, making it an important target for oncology research . Its diverse cellular functions make RANBP10 antibodies valuable tools for investigating multiple biological processes, including cytoskeletal organization, cellular signaling, and disease mechanisms.

How should I optimize Western blot protocols for RANBP10 detection?

For optimal Western blot detection of RANBP10, consider the following methodological approach:

• Sample preparation: Use RIPA buffer supplemented with protease inhibitors to extract total protein from cells or tissues. For platelet-rich samples, add phosphatase inhibitors to prevent protein degradation.

• Gel selection: Due to RANBP10's molecular weight (~90 kDa), use 8-10% SDS-PAGE gels for optimal resolution.

• Transfer conditions: Implement wet transfer at 100V for 60-90 minutes or 30V overnight at 4°C to ensure complete transfer of higher molecular weight proteins.

• Blocking: Use 5% non-fat dry milk in TBST for 1 hour at room temperature to minimize background.

• Primary antibody incubation: Dilute HRP-conjugated RANBP10 antibody according to manufacturer specifications (typically 1:1000-1:5000) and incubate overnight at 4°C .

• Detection: Since the antibody is HRP-conjugated, proceed directly to chemiluminescent detection without secondary antibody. Use enhanced chemiluminescence substrates for optimal sensitivity.

Note that RANBP10 shares extensive protein similarity with its paralog RANBP9, which may complicate detection. Validation using known positive controls (e.g., LNCaP or MCF-7 cell lysates) and negative controls (preferably RANBP10 knockout samples) is essential .

What tissue distribution patterns should I expect when using RANBP10 antibodies?

When performing immunohistochemistry or immunofluorescence with RANBP10 antibodies, researchers should expect the following tissue distribution patterns:

For optimal tissue staining, use antigen retrieval with TE buffer at pH 9.0, followed by appropriate peroxidase blocking if using HRP-conjugated antibodies directly . When examining subcellular localization, expect predominantly cytoplasmic staining with potential enrichment along microtubule structures, particularly in megakaryocytes and platelets. Counterstaining with tubulin markers can help confirm microtubule association and validate antibody specificity.

How can I distinguish between RANBP10 and its paralog RANBP9 in experimental systems?

Distinguishing between RANBP10 and RANBP9 requires careful methodological approaches due to their high sequence similarity:

• Antibody validation: Use knockout/knockdown validation systems. Perform parallel Western blots on RANBP10-/- and RANBP9-/- samples to confirm antibody specificity . The search results describe a specific experiment where RanBP10 knockout/RanBP9-TT double mutant MEFs were used to confirm antibody specificity against RanBP10.

• IP-MS approach: Implement immunoprecipitation followed by mass spectrometry to definitively identify the target protein. This approach can reveal distinctive peptides that differentiate between the paralogs .

• Expression pattern analysis: RANBP10 shows distinctive enrichment in megakaryocytes and platelets, while RANBP9 has broader expression patterns, which can help differentiate in certain tissues .

• Functional validation: Unlike RANBP9, RANBP10 does not interact with Sos and does not activate the Ras pathway, providing a functional distinction between these paralogs .

• Co-IP controls: When performing co-immunoprecipitation experiments, include stringent controls to verify that detected interactions are specific to either RANBP10 or RANBP9 .

To further enhance specificity, consider using epitope-tagged versions of RANBP10 in cellular systems when possible, as demonstrated in the RanBP9-TT mouse model system that allowed for enhanced detection without compromising protein interactions .

What strategies should I employ to investigate RANBP10's role in the CTLH complex?

Investigating RANBP10's role in the CTLH complex requires sophisticated experimental design:

• Co-immunoprecipitation coupled with mass spectrometry: Use HRP-conjugated RANBP10 antibodies to pull down the entire CTLH complex, followed by MS analysis to identify interacting partners. The expected CTLH complex members include Gid8, Muskelin, Maea, Armc8, Wdr26, Rmnd5A/B, Gid4, and Ypel5 .

• Proximity labeling: Implement BioID or APEX2 proximity labeling by fusing these enzymes to RANBP10 to identify proteins in close proximity within living cells, capturing transient interactions within the complex.

• Dynamic interaction analysis: Use FRET or BRET assays to monitor real-time interactions between RANBP10 and other CTLH components under various cellular conditions.

• Domain mapping: Perform systematic truncation analysis of RANBP10 to identify specific domains responsible for CTLH complex incorporation.

• Subcellular fractionation: Separate nuclear and cytoplasmic fractions before immunoprecipitation to better characterize compartment-specific interactions .

When analyzing results, note that all 11 members of the CTLH complex should co-precipitate with RANBP10 (as shown in research where ranking proteins by peptide spectral matches placed all CTLH members within the top 20 hits) . Functional studies should investigate how RANBP10 depletion affects the stability and activity of the entire CTLH complex, potentially revealing its role in the complex's E3 ligase activity.

How can I optimize immunoprecipitation protocols for studying RANBP10 protein interactions?

For optimal immunoprecipitation of RANBP10 and its interacting partners:

• Lysis buffer selection: Use buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, with protease and phosphatase inhibitors. For detecting weaker interactions, consider gentler detergents like 0.3% CHAPS.

• Pre-clearing: Pre-clear lysates with Protein A/G agarose for 1 hour at 4°C to reduce non-specific binding .

• Antibody concentration: For HRP-conjugated RANBP10 antibody, use 5 μg per 1 mg of total protein extract for immunoprecipitation. Alternatively, consider using conjugated resins for direct pull-down .

• Incubation conditions: Perform IP overnight at 4°C with gentle rotation to maximize protein binding while minimizing degradation.

• Washing stringency: Use progressively more stringent washing conditions to remove non-specific interactions while preserving bona fide interactors.

• Cross-linking considerations: For transient interactions, consider using membrane-permeable cross-linkers like DSP to stabilize protein complexes prior to lysis. The search results indicate parallel IP-MS/MS experiments with and without cross-linking were performed for RANBP10 interaction studies .

• Controls: Include proper negative controls such as non-specific IgG and lysates from cells not expressing RANBP10 (or RANBP10 knockout cells when available) .

When analyzing RANBP10 interactors, evaluate against known partners such as PKCγ, PKCδ, and dopamine D1 receptor , as well as CTLH complex components. The interaction with D1 receptor is particularly noteworthy, as it suggests RANBP10's role in receptor phosphorylation and signaling regulation.

How should I design experiments to investigate RANBP10's role in cancer progression?

To investigate RANBP10's role in cancer progression, implement the following experimental design:

• Expression analysis: Quantify RANBP10 expression levels across cancer types using HRP-conjugated RANBP10 antibodies for Western blot, immunohistochemistry, and ELISA. Compare with patient survival data to establish clinical correlations, as has been done for glioblastoma .

• Functional studies:

  • Knockdown/knockout: Use siRNA, shRNA, or CRISPR-Cas9 to deplete RANBP10 in cancer cell lines

  • Overexpression: Generate stable cell lines overexpressing RANBP10 to assess oncogenic potential

  • Phenotypic assays: Measure proliferation, migration, invasion, and apoptosis in modified cells

• Signaling pathway analysis: Investigate RANBP10's impact on:

  • c-Myc stability through FBXW7 regulation, as observed in glioblastoma

  • AR signaling, particularly in prostate cancer models

  • Other oncogenic pathways potentially influenced by RANBP10's GEF activity

• In vivo models: Develop xenograft models using RANBP10-modified cancer cells to assess tumor growth, metastasis, and response to therapies.

• Patient-derived samples: Correlate RANBP10 expression with clinical outcomes, tumor grade, and treatment response in patient-derived specimens.

Research from glioblastoma studies indicates that RANBP10 downregulation significantly inhibits cell proliferation, migration, invasion, and tumor growth . Additionally, RANBP10's ability to suppress FBXW7 promoter activity leads to increased c-Myc protein stability, suggesting a molecular mechanism for its oncogenic effects that should be examined in other cancer types.

What approaches can reveal RANBP10's function in microtubule dynamics and platelet function?

To investigate RANBP10's role in microtubule dynamics and platelet function:

• Live-cell imaging: Utilize fluorescently-tagged tubulin and RANBP10 for real-time visualization of microtubule dynamics in megakaryocytes and platelets. Measure parameters like growth rate, shrinkage rate, catastrophe frequency, and rescue frequency.

• Biochemical assays:

  • Microtubule co-sedimentation assays to assess RANBP10's direct binding to microtubules

  • GTP exchange assays to measure RANBP10's GEF activity toward RAN in the context of microtubule regulation

  • Tubulin polymerization assays with purified components to determine direct effects

• Platelet functional studies:

  • Aggregation assays comparing wild-type and RANBP10-deficient platelets

  • Spreading assays on various substrates to assess morphological changes

  • Clot retraction tests to evaluate contractile force generation

• Ultrastructural analysis:

  • Electron microscopy to examine platelet microtubule marginal band structure

  • Super-resolution microscopy to visualize RANBP10 localization relative to microtubule networks

• In vivo models:

  • Tail bleeding time in RANBP10 knockout mice

  • Thrombus formation under flow conditions

  • Megakaryocyte development and proplatelet formation in bone marrow

Research has shown that RANBP10 localizes to polymerized noncentrosomal microtubules in megakaryocytes and platelets, and its depletion disturbs polymerized filaments in megakaryocytes . RANBP10-null mice exhibited disorders in microtubule filament numbers and localization, indicating its significant role in maintaining microtubule dynamics. These findings provide a foundation for further investigations into how RANBP10 coordinates RAN GTPase activity with cytoskeletal organization in platelets.

How can I address non-specific binding when using RANBP10 antibodies?

Non-specific binding is a common challenge with RANBP10 antibodies due to its similarity with RANBP9. Implement these strategies to improve specificity:

• Antibody validation: Confirm antibody specificity using positive controls (MCF-7 cells, mouse brain tissue, LNCaP cells) and ideally RANBP10 knockout samples as negative controls .

• Blocking optimization:

  • For Western blot: Test both 5% BSA and 5% non-fat dry milk in TBST to determine optimal blocking

  • For IHC/IF: Consider adding 10% normal serum from the same species as the secondary antibody

  • Test longer blocking times (2-3 hours) at room temperature

• Antibody dilution series: Perform a systematic dilution series (1:500 to 1:5000) to identify the optimal concentration that maximizes specific signal while minimizing background.

• Buffer modifications:

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Increase salt concentration (up to 500 mM NaCl) to disrupt low-affinity non-specific binding

  • Add 0.1% SDS to Western blot washing buffers to reduce background

• Pre-adsorption: For critical experiments, consider pre-adsorbing the antibody with recombinant RANBP9 to remove cross-reactive antibodies.

• Alternative detection systems: For HRP-conjugated antibodies showing high background, consider using more sensitive ECL substrates with shorter exposure times.

• Epitope-tagged approaches: When possible, use epitope-tagged RANBP10 expression systems and corresponding tag antibodies, which often provide cleaner results .

The search results note that commercial anti-RANBP10 antibodies often have "poor sensitivity and specificity," highlighting the importance of these optimization steps . When analyzing Western blots, be aware that some RANBP10 antibodies show additional bands at lower molecular weights that may represent degradation products or non-specific binding .

What considerations are important when using RANBP10 antibodies in co-localization studies?

When performing co-localization studies with RANBP10 antibodies:

• Fixation method selection:

  • For preserving microtubule structures: Use 4% paraformaldehyde with 0.1% glutaraldehyde

  • For membrane protein associations: Test methanol fixation at -20°C

  • For nuclear interactions: Compare results with different fixation methods

• Permeabilization optimization:

  • Use 0.1-0.3% Triton X-100 for general permeabilization

  • For delicate structures, consider digitonin (25-50 μg/ml) for selective plasma membrane permeabilization

• Antibody compatibility:

  • Ensure primary antibodies are from different host species to prevent cross-reactivity

  • When using HRP-conjugated RANBP10 antibodies, consider tyramide signal amplification followed by secondary immunofluorescence

• Co-staining markers:

  • Microtubules: Alpha/beta-tubulin for colocalization in megakaryocytes and platelets

  • CTLH complex: Gid8, Muskelin, or Maea as established RANBP10 interactors

  • Membrane receptors: D1 dopamine receptor for receptor signaling studies

• Quantitative analysis:

  • Calculate Pearson's or Mander's correlation coefficients

  • Perform line-scan analysis across subcellular structures

  • Use super-resolution techniques (STED, STORM) for precise localization

• Controls:

  • Single-stained controls to assess bleed-through

  • Competitive blocking with recombinant proteins

  • Knockout/knockdown samples to confirm specificity

Research indicates that RANBP10 localizes to polymerized noncentrosomal microtubules in megakaryocytes and platelets , and also interacts with the D1 dopamine receptor in both kidney and brain tissue . These established localizations serve as positive controls to validate new co-localization findings.

How can I investigate RANBP10's role in receptor signaling pathways?

To study RANBP10's involvement in receptor signaling:

• Receptor phosphorylation analysis:

  • Use phospho-specific antibodies to detect receptor phosphorylation states

  • Implement in vitro kinase assays with purified components

  • Perform phosphorylation site mapping by mass spectrometry

• Signaling cascade investigation:

  • Monitor cAMP accumulation in response to D1 receptor activation with and without RANBP10 overexpression/knockdown

  • Analyze PKC activation patterns in the presence/absence of RANBP10

  • Investigate downstream transcriptional responses using reporter assays

• Protein complex formation:

  • Perform sequential co-immunoprecipitations to identify ternary complexes (e.g., RANBP10-PKC-receptor)

  • Use proximity ligation assays to detect in situ protein interactions

  • Implement split-protein complementation assays to monitor dynamic interactions

• Pharmacological manipulations:

  • Test the effects of PKC inhibitors on RANBP10-mediated receptor phosphorylation

  • Analyze how receptor agonists/antagonists affect RANBP10 recruitment

  • Investigate the impact of GTPase cycle modulators on RANBP10's function

• Trafficking studies:

  • Monitor receptor internalization and recycling in the presence/absence of RANBP10

  • Examine subcellular localization changes upon stimulation

  • Assess receptor half-life and degradation pathways

Research has shown that overexpression of RANBP10 enhances basal D1 receptor phosphorylation, which attenuates D1 receptor-stimulated cAMP accumulation. This increased phosphorylation is blocked by PKC inhibitors, suggesting that RANBP10 regulates receptor phosphorylation by PKC . The data support a model where RANBP10 functions as a signaling integrator, facilitating the efficient regulation of D1 receptor signaling by PKCδ and PKCγ.

What approaches should I use to study the GEF activity of RANBP10 in cellular contexts?

To investigate RANBP10's guanine nucleotide exchange factor (GEF) activity:

• GTP-loading assays:

  • Measure RAN-GTP levels using pull-down assays with RAN-binding domains

  • Implement FRET-based GTP sensors to monitor RAN activation in live cells

  • Quantify GTP/GDP ratios by thin-layer chromatography

• In vitro exchange kinetics:

  • Perform fluorescence-based nucleotide exchange assays with purified components

  • Measure exchange rates under various conditions (pH, temperature, ionic strength)

  • Determine the effects of potential regulatory factors on exchange activity

• Structure-function analysis:

  • Generate point mutations in RANBP10's catalytic domain

  • Create chimeric proteins with other GEF domains

  • Express truncated versions to map minimal GEF activity regions

• Subcellular GEF activity mapping:

  • Use optogenetic approaches to activate RANBP10 in specific cellular compartments

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to assess local GEF activity

  • Measure compartment-specific RAN-GTP/GDP ratios

• Physiological context studies:

  • Investigate how RANBP10's GEF activity affects microtubule dynamics in platelets

  • Examine the impact on nuclear transport in dividing cells

  • Assess effects on mitotic spindle formation

RANBP10 has been established as a cytoplasmic guanine nucleotide exchange factor with activity toward RAN GTPase . This GEF activity is likely crucial for RANBP10's roles in maintaining microtubule dynamics in platelets and megakaryocytes. Given RAN's involvement in nucleocytoplasmic traffic, spindle formation, and nuclear envelope assembly, RANBP10's GEF activity may influence these processes in specific cellular contexts . Understanding the regulation and specificity of this GEF activity is essential for elucidating RANBP10's diverse cellular functions.