VPS50 Antibody

What is VPS50 protein and why is it significant for neuroscience research?

VPS50 (Vacuolar Protein Sorting 50) is a 111.2 kDa protein that functions in synaptic and dense core vesicle acidification . It serves as a component of the EARP complex involved in endocytic recycling . VPS50 is particularly significant for neuroscience research because:

• It is highly expressed in the central nervous system

• Mutations in VPS50 are associated with autism spectrum disorders (ASDs), severe developmental delay, and intellectual disability

• It plays a crucial role in synaptic transmission and plasticity

• Knockout studies in mice demonstrate that VPS50 deletion results in cognitive impairments

The protein's strategic role in vesicle acidification makes it a target of interest for understanding fundamental neuronal processes and neurodevelopmental disorders.

What are the common alternative names and identifiers for VPS50?

Researchers should be aware of several alternative designations when searching literature about VPS50:

Using these alternative designations during literature searches ensures comprehensive coverage of relevant research .

How does VPS50 interact with the V-ATPase complex, and how can antibodies help investigate this relationship?

VPS50 directly interacts with ATP6V1H and appears to facilitate the sorting or assembly of the V-ATPase complex in synaptic vesicles . Research demonstrates that:

• VPS50 proximity to the V-ATPaseV1 domain can be detected using Proximity Ligation Assay (PLA)

• In VPS50 knockout neurons, the PLA signal between VPS50 and V-ATPaseV1 is absent

• VPS50 knockout results in disrupted localization of the V-ATPaseV1 domain

• These disruptions lead to impaired synaptic vesicle acidification

For researchers investigating this interaction, a dual-antibody approach is recommended:

• Use anti-VPS50 antibodies in combination with anti-V-ATPaseV1 antibodies for co-immunoprecipitation

• Employ PLA techniques with these antibody pairs to visualize protein proximity in situ

• Validate antibody specificity with knockout controls to ensure observed interactions are genuine

What evidence supports VPS50's presynaptic versus postsynaptic localization, and how can antibodies confirm this?

Research using proximity ligation assays (PLA) has demonstrated that VPS50 is predominantly presynaptic:

• PLA signal is observed between VPS50 and the presynaptic marker Synapsin1

• No significant PLA signal is detected between VPS50 and the postsynaptic marker PSD95

• Quantification of PLA puncta shows high signal only in the VPS50/Syn1 condition

Researchers can confirm this localization using:

• Immunofluorescence co-localization with established presynaptic markers (Synapsin1, Synaptophysin)

• Subcellular fractionation followed by Western blot analysis

• Immuno-electron microscopy to visualize the ultrastructural localization

When designing these experiments, using rabbit polyclonal VPS50 antibodies with validated specificity is critical for accurate localization studies .

How does VPS50 deletion affect synaptic vesicle properties, and what experimental approaches can detect these changes?

VPS50 knockout studies reveal specific effects on synaptic vesicles that can be investigated with appropriate antibody-based techniques:

• VPS50 KO does not significantly alter the total number of synaptic vesicles (as assessed by electron microscopy)

• Vesicle acidification is substantially reduced in VPS50 KO neurons

• The V-ATPase V1 domain pump is mislocalized in VPS50 KO neurons

• These defects result in impaired synaptic activity

Researchers can detect these changes using:

• The ratio-SyPhy probe (red signal shows all synaptic vesicles; green signal shows vesicles with basic pH)

• Immunofluorescence to track V-ATPase localization

• Electrophysiological recordings to measure functional consequences

• Anti-VPS50 antibodies to confirm knockout efficiency (>70% reduction)

What are the optimal applications and dilutions for VPS50 antibodies in neuronal research?

Based on validation data, the following applications and dilutions are recommended:

For neuronal research specifically, Western blot validation using cerebrum lysate at 40 μg has successfully detected the predicted 111 kDa band . For immunohistochemistry applications, researchers should perform antigen retrieval optimization depending on fixation methods used.

What controls should be included when using VPS50 antibodies in knockout/knockdown studies?

When designing experiments involving VPS50 genetic manipulation, the following controls are essential:

• Genomic validation controls:

  • T7 endonuclease I assay to confirm gene editing efficiency

  • Locus-specific sequencing to verify the edited genomic sequences

• Expression validation controls:

  • qPCR to measure VPS50 mRNA reduction (aim for >70% reduction)

  • Western blot to confirm protein reduction (aim for >70% reduction)

• Functional validation controls:

  • Ratio-SyPhy probe to assess vesicle acidification

  • PLA with V-ATPaseV1 to confirm disruption of protein interactions

Including these controls ensures that observed phenotypes can be confidently attributed to VPS50 loss rather than off-target effects or incomplete knockdown.

How should VPS50 antibodies be stored and handled to maintain optimal performance?

Proper storage and handling are critical for maintaining antibody performance:

Lyophilized antibodies maintain stability at room temperature for at least one month and for greater than a year when kept at -20°C . When reconstituted in sterile pH 7.4 0.01M PBS, the antibody remains stable for at least two weeks at 2-4°C .

How can researchers verify VPS50 antibody specificity for their experimental system?

Validating antibody specificity is crucial for reliable results:

• Knockout/knockdown verification:

  • Test antibodies on CRISPR/Cas9-edited neurons/tissues with confirmed VPS50 deletion

  • Expect >70% reduction in signal intensity in Western blot applications

• Peptide competition assay:

  • Pre-incubate antibody with the immunizing peptide (where known)

  • The specific signal should be significantly reduced or eliminated

• Cross-reactivity testing:

  • If working with non-validated species, perform comparative Western blots

  • Verify that observed molecular weight matches predicted weight (111 kDa)

• Multiple antibody validation:

  • Compare results using antibodies raised against different epitopes

  • Consistent patterns across different antibodies increase confidence in specificity

Some manufacturers have verified specificity using protein arrays containing the target protein plus 383 other non-specific proteins .

What are the key considerations when using VPS50 antibodies for investigating autism spectrum disorder models?

When studying ASD models in relation to VPS50:

• Model selection considerations:

  • Patient-derived mutations can be recapitulated in animal models using CRISPR/Cas9

  • Mosaic knockout in the hippocampus has demonstrated cognitive impairments similar to ASD phenotypes

• Experimental design factors:

  • Assess both structural and functional consequences of VPS50 disruption

  • Combine antibody-based protein detection with behavioral assessments

  • Include age-matched controls to account for developmental effects

• Technical approach:

  • Use stereotaxic AAV delivery for region-specific manipulations

  • Confirm infection efficiency (>90% transduction is achievable)

  • Verify >70% reduction in VPS50 protein using validated antibodies

• Translational considerations:

  • Compare findings with patient data where available

  • Consider both homozygous and heterozygous models to reflect genetic diversity

These approaches can help elucidate mechanisms through which VPS50 mutations contribute to ASD phenotypes and cognitive impairment .

How can VPS50 antibodies be used in combination with synaptic vesicle markers for multiplexed imaging?

For comprehensive analysis of VPS50's role in synaptic function, multiplexed imaging approaches offer powerful insights:

• Recommended antibody combinations:

  • VPS50 (rabbit polyclonal) + Synapsin1 (mouse monoclonal)

  • VPS50 (mouse polyclonal) + V-ATPaseV1 (rabbit polyclonal)

  • VPS50 + ratio-SyPhy probe for simultaneous protein and pH detection

• Imaging optimization:

  • Use secondary antibodies with minimal spectral overlap

  • Consider sequential staining protocols if using multiple primary antibodies from the same host

  • Employ super-resolution microscopy (STED, STORM) for detailed co-localization analysis

• Quantification approaches:

  • Measure co-localization coefficients (Pearson's, Manders')

  • Quantify PLA puncta per field or per defined neuronal compartment

  • Compare signal intensity ratios across experimental conditions

Researchers have successfully used these approaches to demonstrate that VPS50 is near or within synaptic vesicles and proximal to the V-ATPaseV1 domain .

Beyond neuronal systems, what other cellular contexts might benefit from VPS50 antibody-based investigations?

While VPS50 is highly expressed in the brain and skeletal muscle, its role in endocytic recycling suggests broader applications:

• Non-neuronal secretory systems:

  • Endocrine cells that depend on vesicle acidification

  • Immune cells relying on endocytic recycling pathways

  • Kidney tubular cells with active endosomal systems

• Developmental biology:

  • VPS50's association with developmental disorders suggests important roles in embryonic development

  • Investigation of temporal expression patterns during neural development

  • Potential involvement in cell migration and morphogenesis

• Cancer research:

  • Endocytic recycling pathways are often dysregulated in cancer cells

  • VPS50's role in vesicular trafficking may impact drug resistance mechanisms

  • Potential biomarker applications in tumors with aberrant vesicular dynamics

Researchers venturing into these areas should validate antibody performance in each specific tissue context, as expression levels and protein interactions may vary.

What are the technical considerations for using VPS50 antibodies in human patient samples?

Working with human samples requires specific considerations:

• Sample preparation:

  • Post-mortem interval effects on protein degradation should be documented

  • Standardized fixation protocols are critical for immunohistochemistry

  • Consider antigen retrieval optimization for formalin-fixed paraffin-embedded tissues

• Antibody selection:

  • Prioritize antibodies validated specifically for human tissues

  • Both rabbit and mouse host antibodies are available with human reactivity

  • Select antibodies targeting conserved epitopes when comparing with animal models

• Controls and interpretation:

  • Include age-matched control samples

  • Be aware of potential post-translational modifications in disease states

  • Consider genetic variation that might affect epitope recognition

• Ethical and regulatory compliance:

  • Ensure appropriate IRB approval and consent

  • Maintain detailed documentation of antibody validation in human samples

  • Consider patient confidentiality in all reporting

These considerations help ensure reliable and translatable findings when investigating VPS50 in human pathology.