VPS18 Antibody, FITC conjugated

What is VPS18 and why is it significant for research?

VPS18 is a vacuolar protein sorting-associated protein that plays a fundamental role in vesicle-mediated protein trafficking to lysosomal compartments, including the endocytic membrane transport and autophagic pathways. It acts as a core component of the HOPS and CORVET endosomal tethering complexes, which are involved in Rab5-to-Rab7 endosome conversion and regulate membrane fusion events. The significance of VPS18 lies in its critical functions in multiple cellular processes, including autophagy, endosomal trafficking, and signal transduction. Knockout studies in mice have demonstrated that VPS18 deletion leads to severe neurodegeneration, highlighting its importance in maintaining neuronal health and development . Additionally, recent research has uncovered that VPS18 possesses E3 ubiquitin ligase activity, expanding its known functions to include regulation of signaling pathways such as Wnt, estrogen receptor α (ERα), and NFκB .

What are the specifications of the VPS18 Antibody, FITC conjugated?

The VPS18 Antibody, FITC conjugated is a rabbit polyclonal antibody designed for the detection of human VPS18. Its specifications include:

• Host: Rabbit

• Clonality: Polyclonal

• Conjugation: FITC (Fluorescein isothiocyanate)

• Reactivity: Human

• Isotype: IgG

• Purity: >95%

• Purification method: Protein G chromatography

• Form: Liquid

• Buffer composition: 0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% glycerol

• Storage requirements: Aliquot and store at -20°C, avoiding repeated freeze/thaw cycles

These specifications make it suitable for various applications in research settings, including immunofluorescence microscopy for tracking VPS18 localization and function within cells.

What experimental techniques are compatible with this antibody?

Based on the available information, the VPS18 Antibody, FITC conjugated is suitable for several experimental techniques:

• Immunofluorescence microscopy - The FITC conjugation makes this antibody particularly useful for direct visualization of VPS18 localization within cells without requiring a secondary antibody.

• Flow cytometry - The fluorescent tag allows for quantitative analysis of VPS18 expression in cell populations.

• Immunoprecipitation (IP) - Similar antibodies against VPS18 have been validated for IP applications to study protein-protein interactions .

• Western blotting - While the FITC conjugation is primarily beneficial for microscopy applications, the antibody can also be used for Western blot detection of VPS18 protein .

Researchers should note that optimal dilutions/concentrations should be determined experimentally for each specific application and cell type being studied .

How can VPS18 antibodies be used to study its role in the HOPS and CORVET complexes?

The VPS18 antibody, FITC conjugated, provides a valuable tool for investigating VPS18's role in HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering) complexes through multiple approaches:

What methodological considerations should be taken into account when studying VPS18's E3 ubiquitin ligase activity?

Recent research has revealed that VPS18 functions as an E3 ubiquitin ligase, which has significant implications for understanding its role in cellular signaling pathways. When designing experiments to study this activity, researchers should consider:

• RING domain focus: The E3 ligase activity of VPS18 depends on its RING domain. Experiments should incorporate both wild-type VPS18 and RING domain mutants as controls. Previous studies have confirmed the loss of E3 ubiquitin ligase activities for various mutants of VPS18 .

• Ubiquitination target identification: When investigating VPS18's ubiquitination targets, mass spectrometry-based approaches that can detect diGly moieties left after trypsin cleavage of ubiquitinated sites are recommended. This approach has successfully identified multiple proteins whose ubiquitination is affected by VPS18 overexpression .

• Signal pathway analysis: VPS18's E3 ligase activity affects multiple signaling pathways, including Wnt, estrogen receptor α (ERα), and NFκB. Reporter assays for these pathways can help determine how VPS18 manipulations affect downstream signaling. It's important to note that VPS18 affects some pathways (like HIF-1, p53, CREB, and AP-1) in a RING domain-dependent manner, while others (like TGFβ/Smad) are regulated independently of the RING domain .

• Distinguishing between HOPS/CORVET-dependent and independent functions: Careful experimental design is needed to differentiate between VPS18's functions as part of the HOPS/CORVET complexes versus its independent E3 ligase activity. Overexpression studies of individual components can be a better experimental strategy than complex disruption to study specifically VPS18 without disturbing the levels of the HOPS/CORVET complexes .

How can the VPS18 antibody be utilized to investigate neurodegenerative phenotypes?

The VPS18 antibody, FITC conjugated, provides a valuable tool for investigating neurodegenerative phenotypes associated with VPS18 dysfunction:

• Neural migration studies: VPS18 deficiency has been shown to impair neural cell migration in cerebral cortex, hippocampus, and cerebellum. The antibody can be used to track VPS18 expression and localization during neural development, particularly in migration-affected regions like the CA3 region of the hippocampus and Purkinje cells in the cerebellum .

• Autophagosome accumulation detection: Since VPS18 deficiency leads to autophagosome accumulation and blockage of autophagosome clearance, co-staining with the VPS18 antibody and autophagy markers (like LC3) can help visualize this process. In VPS18-deficient neurons, dramatic accumulation of autophagosomes has been observed by electron microscopy, correlating with increased LC3-II levels .

• Ubiquitinated protein aggregation analysis: VPS18 knockout mice show accumulation of ubiquitinated proteins and formation of inclusion bodies positive for p62. The VPS18 antibody can be used alongside ubiquitin and p62 antibodies to investigate how VPS18 dysfunction leads to protein aggregation, which is a common feature of many neurodegenerative diseases .

• Apoptosis correlation studies: VPS18 deficiency triggers neuronal apoptosis, as evidenced by increased caspase-3 activation. Researchers can use the VPS18 antibody together with apoptosis markers to investigate the relationship between VPS18 expression levels and neuronal survival, potentially identifying thresholds of VPS18 function needed to prevent neurodegeneration .

What are the optimal fixation and permeabilization protocols for immunofluorescence with FITC-conjugated VPS18 antibody?

When conducting immunofluorescence with the FITC-conjugated VPS18 antibody, researchers should consider the following protocol optimizations:

• Fixation method selection:

  • For preserving membrane structures (critical for studying endosomal compartments): 4% paraformaldehyde (PFA) in PBS for 15-20 minutes at room temperature is recommended.

  • For better antigen accessibility: Methanol fixation (-20°C for 10 minutes) may be preferable as it simultaneously fixes and permeabilizes cells.

• Permeabilization considerations:

  • For PFA-fixed samples: Use 0.1-0.2% Triton X-100 in PBS for 5-10 minutes to permeabilize membranes without disrupting endosomal structures.

  • For studying fine membrane interactions: Consider milder detergents like 0.1% saponin, which maintains membrane integrity better than Triton X-100.

• Blocking protocol:

  • Block with 5% normal serum (matched to the host species of any other primary antibodies being used) and 1% BSA in PBS for 30-60 minutes to reduce background.

  • Include 0.1% Triton X-100 in the blocking solution if permeabilization is needed.

• Antigen retrieval considerations:

  • For tissue sections or difficult-to-access epitopes: Consider citrate buffer (pH 6.0) heat-mediated antigen retrieval.

• Important controls:

  • Include a negative control using an isotype-matched rabbit IgG-FITC conjugate to assess non-specific binding.

  • When performing co-localization studies, include single-stained controls to account for potential spectral overlap.

How should experimental design address the dual roles of VPS18 in trafficking and ubiquitination?

To effectively study the complex roles of VPS18 in both vesicular trafficking and protein ubiquitination, researchers should implement an integrated experimental approach:

• Separation of function experimental design:

  • Compare wild-type VPS18 with RING domain mutants that specifically abolish E3 ligase activity but maintain trafficking functions. This allows researchers to attribute observed phenotypes to either ubiquitination or trafficking roles .

  • Utilize truncation mutants that selectively disrupt interactions with either HOPS/CORVET components or ubiquitination machinery.

• Complementary assays for different functions:

  • For trafficking function: Monitor endosomal fusion events, autophagosome clearance, and lysosomal delivery using fluorescent reporters (e.g., mCherry-GFP-LC3 for autophagy flux).

  • For ubiquitination activity: Implement ubiquitination assays with purified components in vitro, or cell-based ubiquitination detection systems.

• Time-resolved analysis:

  • Use pulse-chase approaches to distinguish between immediate effects (likely E3 ligase activity) and delayed effects (potentially related to altered trafficking).

  • Consider inducible expression systems to control the timing of VPS18 manipulation.

• Context-specific investigations:

  • Examine how different cellular stresses (starvation, oxidative stress, etc.) might shift the balance between VPS18's trafficking and ubiquitination functions.

  • Assess how these functions may be coordinated in specific cellular processes like autophagy or endocytosis.

• Interaction partner analysis:

  • Use the VPS18 antibody for co-immunoprecipitation studies to identify interaction partners under different conditions.

  • Compare interactomes when cells are manipulated to enhance either trafficking or ubiquitination functions.

What technical considerations are important when using the VPS18 antibody for co-localization studies?

When conducting co-localization studies with the FITC-conjugated VPS18 antibody, several technical considerations are crucial for obtaining reliable and interpretable results:

• Fluorophore selection and spectral considerations:

  • FITC emits in the green spectrum (peak ~525 nm), so choose complementary fluorophores like TRITC (red) or Cy5 (far-red) for co-staining to minimize spectral overlap.

  • If using multiple fluorophores, perform appropriate controls to account for bleed-through and cross-talk between channels.

• Microscopy and image acquisition parameters:

  • Use confocal microscopy rather than widefield to achieve better spatial resolution for endosomal structures.

  • Maintain consistent exposure settings between experimental conditions.

  • Consider super-resolution techniques (STED, STORM, etc.) for detailed analysis of VPS18's association with endosomal subdomains.

• Fixation timing considerations:

  • Since VPS18 participates in dynamic trafficking processes, the timing of fixation can significantly impact observed localization patterns.

  • Consider time-course experiments to capture different stages of endosomal maturation or autophagy.

• Quantitative co-localization analysis:

  • Use appropriate software (ImageJ with Coloc2, CellProfiler, etc.) to quantify co-localization.

  • Report multiple co-localization metrics (Pearson's correlation coefficient, Mander's overlap coefficient) for comprehensive analysis.

  • Perform statistical analysis on multiple cells and across independent experiments.

• Biological controls for specificity:

  • Include VPS18 knockdown or knockout samples as negative controls to confirm antibody specificity.

  • Use known markers of endosomal compartments (Rab5, Rab7, LAMP1) as reference points for VPS18 localization.

How can researchers distinguish between VPS18's role in autophagy versus endosomal trafficking?

Distinguishing between VPS18's functions in autophagy and endosomal trafficking requires careful experimental design and specific markers:

• Dual-reporter systems:

  • Use tandem fluorescent-tagged reporters like mRFP-GFP-LC3 to monitor autophagy flux. In this system, GFP fluorescence is quenched in acidic environments while mRFP remains stable, allowing researchers to distinguish between autophagosomes (yellow = GFP+/mRFP+) and autolysosomes (red = GFP-/mRFP+).

  • Compare this with endocytic cargo tracking (e.g., fluorescently-labeled transferrin or EGF) to separate effects on different pathways.

• Pathway-specific manipulations:

  • Combine VPS18 antibody staining with treatments that specifically induce autophagy (starvation, rapamycin) or block it at different stages (bafilomycin A1 to prevent acidification).

  • Compare results with manipulations of endocytosis (e.g., dynamin inhibitors, low temperature blocks).

• Quantitative assessment strategies:

  • For autophagy: Measure LC3-II/LC3-I ratios, p62 accumulation, and autophagic vesicle counts. In VPS18-deficient neurons, both LC3-II and LC3-I levels increase, and p62-positive inclusions form, indicating blocked autophagosome clearance .

  • For endocytosis: Track rates of internalization and degradation of model cargoes (e.g., EGFR, transferrin receptor).

• Ultrastructural analysis:

  • Electron microscopy can differentiate between accumulated autophagosomes (double-membrane structures containing cytoplasmic material) and endosomal compartments (single-membrane structures). VPS18-deficient neurons show dramatic accumulation of autophagosomes .

  • Immunogold labeling with the VPS18 antibody can pinpoint its exact localization on specific vesicular structures.

• Temporal considerations:

  • Early effects of VPS18 manipulation likely reflect its primary functions, while later effects may represent compensatory responses or secondary consequences.

What are common pitfalls in data interpretation when studying VPS18 signaling pathways?

When investigating VPS18's role in signaling pathways, researchers should be aware of several potential pitfalls in data interpretation:

How can researchers address contradictory results between in vitro and in vivo studies of VPS18?

Reconciling contradictory results between in vitro and in vivo studies of VPS18 requires systematic approaches to bridge these experimental contexts:

• Context-specific function analysis:

  • VPS18 may have different priorities in distinct cellular environments. For example, its role in neural cell migration is particularly prominent in the cerebellum compared to cerebral cortex .

  • Approach: Use tissue-specific or cell-type-specific conditional knockout models rather than global knockouts to pinpoint context-dependent functions.

• Developmental timing considerations:

  • In vivo studies in mice show that VPS18 is critical for proper neuronal migration and survival during development , which may not be apparent in established cell lines.

  • Approach: Use developmental model systems (embryoid bodies, organoids) that better recapitulate the developmental context while maintaining some experimental control.

• Compensation mechanism identification:

  • Acute disruption in vitro may show different phenotypes than chronic loss in vivo due to compensatory adaptations.

  • Approach: Use inducible systems for both in vitro and in vivo models to compare acute versus chronic effects of VPS18 loss or mutation.

• Multi-level analysis strategy:

  • Combine biochemical (protein levels, ubiquitination status), cellular (trafficking, localization), and physiological (neuronal function, migration) readouts across experimental systems.

  • Approach: Establish a core set of assays that can be performed both in vitro and in vivo to create directly comparable datasets.

• Translational validation approach:

  • When in vitro findings suggest potential mechanisms, validate these in vivo using targeted approaches.

  • Example: If in vitro studies identify specific ubiquitination targets of VPS18, confirm the relevance of these modifications in vivo by creating non-ubiquitinatable mutants of the target proteins in mouse models.

What emerging research areas might benefit from VPS18 antibody applications?

The FITC-conjugated VPS18 antibody holds significant potential for several emerging research areas:

• Neurodegenerative disease mechanisms:

  • Given VPS18's critical role in preventing neurodegeneration , this antibody could help investigate how defects in endolysosomal trafficking contribute to diseases like Alzheimer's, Parkinson's, and ALS.

  • Potential application: Examining how disease-associated proteins interact with VPS18-positive compartments and whether VPS18 dysfunction contributes to protein aggregation.

• Cancer biology:

  • The newly discovered role of VPS18 as an E3 ubiquitin ligase affecting signaling pathways like Wnt and ERα suggests potential implications in cancer development and progression .

  • Potential application: Investigating VPS18 expression and localization in tumor samples and correlating with pathway activation status.

• Developmental neurobiology:

  • VPS18's role in neuronal migration makes it relevant for studying neurodevelopmental disorders.

  • Potential application: Using the antibody to track VPS18 dynamics during critical periods of brain development in various model systems.

• Autophagy regulation in aging:

  • Since VPS18 is crucial for autophagosome clearance , and autophagy dysfunction is implicated in aging, the antibody could help investigate age-related changes in VPS18 function.

  • Potential application: Comparing VPS18 localization and function in tissues from young versus aged organisms.

• Cross-talk between vesicular trafficking and signaling:

  • The dual roles of VPS18 in trafficking and signaling pathway regulation position it as a model protein for studying how these cellular processes interact.

  • Potential application: Using the antibody for proximity labeling approaches to identify novel VPS18 interaction partners under different signaling conditions.

What methodological advances could enhance the utility of VPS18 antibodies in research?

Several methodological advances could significantly enhance the research utility of VPS18 antibodies:

• Super-resolution microscopy applications:

  • Adapting the FITC-conjugated VPS18 antibody for super-resolution techniques like STORM, PALM, or STED could reveal previously undetectable spatial relationships between VPS18 and other proteins within endosomal subdomains.

  • This approach could help resolve how VPS18 organizes within HOPS and CORVET complexes at the nanometer scale.

• Live-cell imaging adaptations:

  • Developing membrane-permeable VPS18 nanobodies conjugated to fluorophores could enable real-time tracking of endogenous VPS18 in living cells.

  • This would allow visualization of dynamic VPS18 behavior during vesicle fusion events or complex assembly.

• Proximity labeling integration:

  • Combining VPS18 antibodies with proximity labeling techniques (BioID, APEX) could identify transient or weak interactors in different cellular compartments.

  • This approach could help map the local proteome around VPS18 under different conditions, potentially identifying novel substrates for its E3 ligase activity.

• Single-molecule tracking:

  • Using quantum dot-conjugated antibody fragments to track individual VPS18 molecules could reveal how its dynamics change during endosome maturation or in response to signaling events.

• Cryo-electron tomography:

  • Immunogold labeling with VPS18 antibodies for cryo-electron tomography could provide structural insights into how VPS18 organizes tethering complexes at membrane contact sites.

  • This approach could bridge biochemical data with structural understanding of VPS18 function.

What are the most promising therapeutic implications of VPS18 research?

Research into VPS18 functions has revealed several promising therapeutic implications:

• Neuroprotective strategies:

  • Given that VPS18 deletion leads to severe neurodegeneration , enhancing VPS18 function or compensating for its loss in at-risk neurons could potentially prevent or slow neurodegeneration.

  • Approach: Developing small molecules that enhance the efficiency of remaining VPS18 or that can bypass VPS18-dependent steps in autophagosome clearance.

• Targeting VPS18's E3 ligase activity:

  • The discovery that VPS18 functions as an E3 ubiquitin ligase regulating signaling pathways opens possibilities for modulating these pathways in disease .

  • Approach: Developing selective inhibitors or activators of VPS18's E3 ligase activity without affecting its trafficking functions, potentially allowing pathway-specific interventions.

• Neuronal migration disorder treatments:

  • VPS18's role in neuronal migration suggests potential applications in addressing neurodevelopmental disorders associated with improper neuronal positioning.

  • Approach: Identifying the specific mechanisms by which VPS18 influences migration could reveal targetable downstream effectors.

• Cancer therapy applications:

  • If VPS18's regulation of signaling pathways like Wnt and ERα proves important in cancer development or progression, it could represent a novel therapeutic target.

  • Approach: Investigating whether VPS18 inhibition could synergize with existing therapies targeting these pathways.

• Enhanced lysosomal function in lysosomal storage disorders:

  • Since VPS18 is critical for proper lysosomal function through its role in membrane fusion, modulating its activity might benefit conditions characterized by lysosomal dysfunction.

  • Approach: Exploring whether VPS18 enhancement could improve clearance of accumulated substrates in lysosomal storage disorders.