VPS28 Antibody, HRP conjugated

What is VPS28 and why is it important in cellular research?

VPS28 (Vacuolar protein sorting-associated protein 28 homolog) functions as a subunit of the ESCRT-I complex (endosomal sorting complex I required for transport), which mediates the transport and sorting of proteins into subcellular vesicles. This 25 kDa protein plays a critical role in regulating vesicular trafficking processes and interacts with VPS23 (also known as TSG101) . Recent research has revealed that VPS28 is highly expressed in neurons and is involved in the secretion of neuronal extracellular vesicles (EVs). Furthermore, it participates in the formation of multivesicular bodies (MVBs) and plays an important role in VEGF-A transport, promoting neurovascular communication . The protein's involvement in these fundamental cellular processes makes it a valuable target for research in cell biology, neuroscience, and vascular biology.

What sample types have been validated for VPS28 antibody applications?

VPS28 antibodies have been successfully validated with multiple sample types across species. Western blot applications have confirmed reactivity with:

• Human cell lines: HepG2, Jurkat, and A549 cells

• Human tissue: fetal brain and fetal liver

• Mouse brain tissue

• Rat brain tissue

For immunofluorescence/immunocytochemistry (IF/ICC) applications, positive results have been observed in:

• HepG2 cells

Immunohistochemistry analysis has been successful with:

• Paraffin-embedded human kidney tissue

This cross-species reactivity makes VPS28 antibodies versatile tools for comparative studies across human, mouse, and rat models.

What applications are validated for detecting VPS28?

VPS28 antibodies have been validated for multiple research applications:

For optimal results, it is recommended to titrate these antibodies in each testing system, as effectiveness may be sample-dependent .

How should VPS28 antibodies be stored and handled for optimal performance?

For optimal longevity and performance, VPS28 antibodies should be stored at -20°C, where they remain stable for one year after shipment . The antibodies are typically supplied in storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, for -20°C storage, aliquoting is generally unnecessary, which simplifies handling. Some preparations may contain 0.1% BSA for added stability . When working with the antibody, avoid repeated freeze-thaw cycles and keep the antibody on ice during experiments to preserve activity. Before use, allow the antibody to equilibrate to room temperature and gently mix to ensure homogeneity without introducing bubbles or denaturing the protein.

How can VPS28 antibodies be applied to study ESCRT-I complex dynamics?

To study ESCRT-I complex dynamics using VPS28 antibodies, researchers can employ multiple complementary approaches. Co-immunoprecipitation using anti-VPS28 antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate) can pull down the entire ESCRT-I complex, allowing identification of interacting partners through subsequent Western blot or mass spectrometry analysis . For visualization of complex formation, dual immunofluorescence combining VPS28 antibodies with antibodies against other ESCRT-I components (VPS23/TSG101, VPS37, and MVB12A/B) can reveal co-localization patterns within cells .

Importantly, dysfunction in VPS28 dramatically affects multivesicular body (MVB) formation. Research has shown that VPS28 knockdown results in clustered localization of HGS (an MVB marker) and significant reduction in the number and density of MVBs, as visualized by transmission electron microscopy . For dynamic studies, researchers can employ live-cell imaging of fluorescently tagged VPS28 combined with fixed-cell immunolabeling using VPS28 antibodies to validate observed patterns. This multi-method approach provides comprehensive insights into how VPS28 contributes to ESCRT-I complex assembly, localization, and function within the endosomal pathway.

What methodologies can be employed to study VPS28's role in extracellular vesicle (EV) secretion?

To investigate VPS28's role in EV secretion, researchers should adopt a multi-faceted approach. Western blot analysis using VPS28 antibodies (1:500-1:1000 dilution) can assess the protein's expression in both cells and isolated EVs . For EV isolation, differential ultracentrifugation followed by analysis of EV markers CD63 and TSG101 alongside VPS28 is recommended . Research has demonstrated that VPS28 knockdown significantly decreases TSG101 levels in ultracentrifuged EV pellets and drastically reduces CD63 levels in secreted EVs, the 2K pellet, and whole cell lysates .

For quantitative analysis, nanoparticle tracking analysis (NTA) can measure changes in EV numbers upon VPS28 manipulation. In zebrafish models, VPS28 knockout resulted in reduced EV secretion compared to controls . To visualize EV biogenesis, researchers can employ confocal microscopy on cells expressing CD63-GFP constructs, as VPS28 mutants show significantly decreased numbers of GFP-positive endosomes . For functional studies, isolated EVs from control and VPS28-depleted cells can be applied to recipient cells to assess their biological effects, particularly on angiogenesis through VEGF-A trafficking. This comprehensive approach enables quantitative and qualitative assessment of how VPS28 regulates EV biogenesis, secretion, and function.

How can researchers investigate VPS28's role in neurovascular development?

To investigate VPS28's role in neurovascular development, researchers should implement a strategic combination of in vivo and in vitro approaches. In zebrafish models, VPS28 has been found to be abundantly expressed in the central nervous system (CNS) at 24 and 48 hours post-fertilization (hpf), with GFP reporter constructs under the VPS28 promoter showing specific expression in the brain . Flow cytometry and real-time PCR analyses have confirmed that VPS28 is more enriched in neurons than in endothelial cells .

For functional studies, researchers can use VPS28 antibodies (1:50-1:500 dilution for immunofluorescence) to examine protein expression and localization in neural and vascular tissues . Knockout or knockdown approaches followed by rescue experiments with neuron-specific expression can determine tissue-specific requirements, as neuronal-derived VPS28 has been shown to rescue vascular phenotypes . Co-culture systems with neurons and endothelial cells, where neurons are manipulated for VPS28 expression, can help assess paracrine effects on endothelial behavior.

To specifically investigate VEGF-A trafficking, researchers should examine VEGF-A content in EVs from control and VPS28-depleted neurons using appropriate antibodies. This multi-layered approach provides comprehensive insights into how neuronal VPS28 influences vascular development through regulation of EV secretion and VEGF-A trafficking.

What are the recommended protocols for detecting VPS28 in immunohistochemistry?

For optimal detection of VPS28 in immunohistochemistry (IHC) of paraffin-embedded tissues, the following protocol is recommended:

• Tissue preparation: Properly fix tissues in formalin and embed in paraffin following standard procedures.

• Sectioning: Cut tissues into 4-6 µm sections and mount on positively charged slides.

• Deparaffinization and rehydration: Process slides through xylene and graded alcohols following standard protocols.

• Antigen retrieval: Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol, as this specific condition has been validated for VPS28 detection .

• Blocking: Block endogenous peroxidase activity with 3% hydrogen peroxide, followed by protein blocking with appropriate serum.

• Primary antibody incubation: Apply VPS28 antibody at 1:50 dilution and incubate at 4°C overnight in a humidified chamber.

• Detection system: Apply appropriate secondary antibody conjugated to HRP or biotin, followed by signal amplification if needed.

• Visualization: Develop signal using DAB substrate and counterstain with hematoxylin.

• Mounting: Dehydrate sections, clear in xylene, and mount with permanent mounting medium.

This protocol has been successfully applied for VPS28 detection in human kidney tissue and can be adapted for other tissue types with appropriate optimization.

How can I validate VPS28 antibody specificity in knockout/knockdown systems?

To rigorously validate VPS28 antibody specificity, implement a comprehensive knockout/knockdown validation strategy:

• siRNA knockdown: Transfect cells with VPS28-targeted siRNA and confirm knockdown efficiency via Western blot. This approach has been successfully employed in HEK293T cells, where VPS28 siRNA knockdown showed clear reduction in protein levels compared to control siRNA .

• CRISPR/Cas9 knockout: Generate complete VPS28 knockout cell lines for the strongest validation. When probing with the VPS28 antibody, the specific band at 25-30 kDa should be absent in knockout samples.

• Genetic models: Utilize existing VPS28 mutant animal models, such as the zebrafish VPS28 mutant line, for tissue validation .

• Multiple antibody comparison: Test different VPS28 antibodies targeting distinct epitopes to ensure consistent results. The commercially available antibodies from different sources (e.g., ab167172, 15478-1-AP) provide this opportunity .

• Western blot analysis: Run control and knockout/knockdown samples side-by-side, looking for the disappearance of the specific band at 25-30 kDa. Include positive control samples such as Jurkat cells, HepG2 cells, or brain tissue which have confirmed VPS28 expression .

• Immunofluorescence validation: Perform parallel staining in control and knockout/knockdown cells to visualize loss of specific signal.

This multi-faceted approach ensures that observed signals truly represent VPS28 protein, minimizing the risk of misinterpreting non-specific binding or cross-reactivity.

How can I troubleshoot weak or absent signals in VPS28 Western blots?

When experiencing weak or absent signals in VPS28 Western blots, consider the following systematic troubleshooting approach:

• Sample preparation: Ensure complete cell lysis using buffers containing appropriate detergents. VPS28 is associated with membrane structures, so inadequate solubilization may result in protein loss during sample preparation.

• Protein concentration: Increase sample loading to 10-20 μg protein per lane, as validated protocols have successfully used 10 μg of cell lysate (HepG2, Jurkat, A549) or tissue lysate (human fetal brain, human fetal liver) .

• Antibody concentration: If signal remains weak, increase primary antibody concentration. Try a range between 1:250 to 1:1000 instead of the standard 1:1000 dilution .

• Incubation conditions: Extend primary antibody incubation to overnight at 4°C to improve binding.

• Detection system: Ensure secondary antibody compatibility (e.g., goat anti-rabbit HRP at 1:2000 dilution for rabbit primary antibodies) . Consider using enhanced chemiluminescence (ECL) substrate with higher sensitivity.

• Membrane optimization: PVDF membranes may provide better protein retention than nitrocellulose for some applications.

• Positive controls: Include known positive samples such as Jurkat cells, HepG2 cells, or brain tissue lysate alongside experimental samples .

• Transfer efficiency: Verify protein transfer using reversible staining methods like Ponceau S before immunoblotting.

This systematic approach should help identify and resolve technical issues causing weak VPS28 signals in Western blot applications.

What controls are essential when using VPS28 antibodies in research experiments?

Implementing appropriate controls is crucial for generating reliable and interpretable results with VPS28 antibodies:

• Positive tissue/cell controls: Include samples known to express VPS28, such as:

  • Human: HepG2, Jurkat, or A549 cells

  • Mouse/Rat: Brain tissue
    These validated samples ensure the antibody detection system is working properly.

• Negative controls: For experiments where tissue-specific expression is important, include samples with low or no VPS28 expression. Based on expression patterns, non-CNS tissues at certain developmental stages may serve as negative controls .

• Primary antibody omission control: Process samples identically but omit the primary VPS28 antibody to identify non-specific secondary antibody binding.

• Isotype control: Use non-specific IgG from the same host species and at the same concentration as the VPS28 antibody to identify non-specific binding.

• Knockdown/knockout validation: When possible, include VPS28 knockdown or knockout samples to confirm antibody specificity. This approach has been demonstrated with siRNA in HEK293T cells .

• Loading controls: For Western blots, always include appropriate loading controls (β-actin, GAPDH, etc.) to normalize VPS28 expression.

• Molecular weight marker: Always run a molecular weight marker to confirm that the observed band corresponds to the expected size of VPS28 (calculated 25 kDa, observed 28-30 kDa) .

These comprehensive controls ensure experimental rigor and facilitate accurate interpretation of results when working with VPS28 antibodies.

What are the optimal tissue fixation methods for immunohistochemical detection of VPS28?

For optimal immunohistochemical detection of VPS28 in tissue sections, the following fixation and processing approaches are recommended:

• Fixative selection: 10% neutral buffered formalin is the recommended fixative for tissues intended for VPS28 immunostaining, as successful detection has been demonstrated in formalin-fixed, paraffin-embedded human kidney tissue .

• Fixation duration: Limit fixation time to 24-48 hours to prevent excessive cross-linking that might mask VPS28 epitopes. Over-fixation can significantly reduce antibody binding and signal intensity.

• Antigen retrieval: Heat-mediated antigen retrieval using citrate buffer (pH 6.0) is critical for VPS28 detection in paraffin sections. This specific condition has been validated and should be performed before commencing with the IHC staining protocol .

• Frozen sections alternative: For tissues where paraffin embedding causes excessive antigen masking, consider using frozen sections fixed briefly with 4% paraformaldehyde, followed by permeabilization with 0.1-0.2% Triton X-100.

• Cell culture fixation: For cultured cells in immunocytochemistry applications, 4% paraformaldehyde fixation for 15 minutes at room temperature followed by permeabilization with 0.1% Triton X-100 provides good results for VPS28 detection in HepG2 cells .

• Pre-embedding procedures: Ensure tissue dehydration, clearing, and paraffin infiltration steps are not prolonged beyond necessary times, as extended processing can contribute to antigen masking.

Following these validated fixation and processing methods will help preserve VPS28 antigenicity and ensure optimal immunohistochemical detection.

How can researchers quantify VPS28 expression changes in experimental conditions?

For accurate quantification of VPS28 expression changes under experimental conditions, researchers should employ multiple complementary approaches:

• Western blot densitometry:

  • Run VPS28 Western blots using antibody dilutions of 1:500-1:1000

  • Capture digital images within the linear dynamic range of detection

  • Quantify band intensity using software like ImageJ or specialized gel analysis programs

  • Normalize VPS28 signal to appropriate loading controls (GAPDH, β-actin, tubulin)

  • Run technical replicates (n≥3) across multiple biological samples for statistical validity

• qRT-PCR for mRNA quantification:

  • Design primers specific to VPS28 mRNA sequences

  • Validate primer efficiency using standard curves

  • Normalize to multiple reference genes for accuracy

  • Compare protein and mRNA levels to identify potential post-transcriptional regulation

• Immunofluorescence quantification:

  • Use consistent antibody dilutions (1:50-1:500 for IF/ICC)

  • Capture images using identical microscope settings

  • Analyze fluorescence intensity per cell using automated image analysis software

  • Measure intensities in specific subcellular compartments to detect redistribution

• Flow cytometry (for compatible samples):

  • Perform intracellular staining for VPS28 following appropriate fixation and permeabilization

  • Use flow cytometry to quantify VPS28 expression at the single-cell level

  • Apply appropriate gating strategies to examine expression in specific cell populations

• Controls for quantification:

  • Include gradient dilutions of positive control samples to ensure measurements fall within linear range

  • Process experimental and control samples simultaneously to minimize technical variation

  • Include VPS28 knockdown/knockout samples as negative controls for signal specificity validation

This multi-method approach provides robust quantification of VPS28 expression changes while controlling for potential artifacts from any single method.

What are the considerations for multiplexing VPS28 antibodies with other markers?

When multiplexing VPS28 antibodies with other markers for co-localization studies, researchers should consider these critical factors:

• Antibody compatibility: Select primary antibodies raised in different host species to avoid cross-reactivity. If using the rabbit anti-VPS28 antibodies (ab167172, 15478-1-AP) , pair with mouse, rat, or goat antibodies against other targets.

• Epitope masking: When studying ESCRT-I complex components, be aware that protein-protein interactions may mask epitopes. Sequential staining or proximity ligation assays may be required if standard co-staining fails.

• Fluorophore selection: Choose fluorophores with minimal spectral overlap when designing multiplexed immunofluorescence experiments. For VPS28 co-localization with endosomal markers, consider these validated combinations:

  • VPS28 + HGS (MVB marker): demonstrated in VPS28 knockdown studies

  • VPS28 + CD63 (EV marker): successful visualization in neuronal expression studies

• Signal amplification: For low-abundance proteins, coordinate amplification systems (tyramide signal amplification, antibody-oligonucleotide conjugates) to maintain balanced signal intensities across markers.

• Subcellular localization optimization: When examining VPS28 co-localization with endosomal markers, optimize permeabilization conditions—mild detergents (0.1% Triton X-100 or 0.1% saponin) typically provide good access to endosomal structures while preserving morphology.

• Validation controls: Include single-stained samples and fluorescence-minus-one controls to confirm specificity of co-localization patterns. Additionally, use VPS28 knockout/knockdown samples as negative controls to confirm staining specificity .

• Image acquisition settings: Use sequential scanning on confocal microscopes to eliminate crosstalk between channels, particularly important when examining small structures like endosomes and multivesicular bodies where VPS28 functions.

Following these guidelines will enable high-quality multiplexed detection of VPS28 alongside other cellular markers for comprehensive analysis of ESCRT-I function and endosomal dynamics.

How should researchers design experiments to study VPS28's role in multivesicular body formation?

To effectively study VPS28's role in multivesicular body (MVB) formation, researchers should implement the following experimental design:

• Genetic manipulation strategies:

  • siRNA knockdown: Transfect cells with VPS28-targeted siRNA and confirm knockdown efficiency via Western blot using VPS28 antibodies at 1:500-1:1000 dilution

  • CRISPR/Cas9 knockout: Generate complete VPS28 knockout cell lines for more dramatic phenotypes

  • Rescue experiments: Re-express VPS28 in knockout cells to confirm phenotype specificity

  • Dominant-negative approaches: Express truncated VPS28 variants that disrupt ESCRT-I assembly

• MVB visualization techniques:

  • Immunofluorescence: Employ VPS28 antibodies (1:50-1:500 dilution) alongside established MVB markers such as HGS

  • Electron microscopy: Use transmission electron microscopy (TEM) to directly visualize and quantify MVB morphology, as successfully employed in VPS28 knockdown studies

  • Live-cell imaging: Transfect cells with fluorescently tagged endosomal markers and perform time-lapse imaging

• Functional assays:

  • Rab5(Q79L) assay: Transfect cells with the GTPase-defective Rab5(Q79L) mutant to form enlarged endosomes and assess VPS28's impact on endosome morphology and number

  • Cargo sorting assays: Track the endosomal sorting of known ESCRT-dependent cargo proteins in VPS28-depleted cells

  • EV isolation and characterization: Isolate EVs using ultracentrifugation and analyze marker proteins (CD63, TSG101) via Western blot to assess MVB-to-EV transition

• Quantitative analysis:

  • Measure MVB size, number, and morphology in control versus VPS28-depleted cells

  • Quantify co-localization coefficients between VPS28 and other endosomal markers

  • Perform Western blot analysis of MVB markers in different subcellular fractions

This comprehensive experimental approach, combining genetic, microscopic, and biochemical methods, will provide robust insights into VPS28's specific contributions to MVB formation and function.

What are the recommended protocols for co-immunoprecipitation studies with VPS28 antibodies?

For successful co-immunoprecipitation (co-IP) studies with VPS28 antibodies to investigate ESCRT-I complex interactions, follow this optimized protocol:

• Cell lysis and sample preparation:

  • Harvest cells at 80-90% confluence (HepG2, Jurkat, or other validated cell types)

  • Lyse cells in ice-cold IP lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate plus protease inhibitors)

  • Clear lysate by centrifugation at 14,000 g for 10 minutes at 4°C

  • Pre-clear lysate with Protein A/G beads for 1 hour at 4°C

• Antibody binding:

  • Use 0.5-4.0 μg of VPS28 antibody per 1.0-3.0 mg of total protein lysate, as validated for mouse brain tissue samples

  • Incubate pre-cleared lysate with VPS28 antibody overnight at 4°C with gentle rotation

  • Add 30-50 μL of Protein A/G beads and incubate for 2-4 hours at 4°C

• Washing and elution:

  • Wash beads 4-5 times with washing buffer (lysis buffer with reduced detergent concentration)

  • Elute protein complexes by boiling in SDS sample buffer for 5 minutes at 95°C

• Analysis of co-immunoprecipitated proteins:

  • Separate proteins by SDS-PAGE and perform Western blot analysis

  • Probe for VPS28 (25-30 kDa) to confirm successful IP

  • Probe for known ESCRT-I components: VPS23/TSG101, VPS37, and MVB12A/B

  • Probe for potential novel interacting partners

• Controls:

  • Input control: 5-10% of pre-cleared lysate

  • IgG control: Parallel IP using non-specific IgG from the same species as the VPS28 antibody

  • Negative control: Lysate from VPS28 knockout or knockdown cells to verify antibody specificity

• Reverse co-IP:

  • Perform parallel co-IP using antibodies against suspected interacting partners

  • Probe Western blots for VPS28 to confirm bidirectional interaction

This protocol has been optimized based on successful VPS28 immunoprecipitation from mouse brain tissue and can be adapted for other sample types with appropriate optimization.

How can I optimize immunofluorescence protocols for detecting VPS28 in neuronal cells?

To optimize immunofluorescence protocols for detecting VPS28 in neuronal cells, where the protein is highly expressed and plays critical functional roles , follow these specialized procedures:

• Primary neuronal culture preparation:

  • For mouse cortical neurons, seed cells at moderate density (75,000-100,000 cells/cm²) on poly-L-lysine coated coverslips

  • Allow neurons to mature appropriately (7-14 days in vitro) before fixation, as VPS28 expression has been confirmed in mature mouse cortical neurons (Tubb3+)

• Fixation and permeabilization optimization:

  • Fix neurons with 4% paraformaldehyde for 15 minutes at room temperature

  • For optimal endosomal visualization, use mild permeabilization with 0.1% Triton X-100 for 10 minutes

  • Alternative gentle permeabilization with 0.1% saponin may better preserve membrane structures where VPS28 localizes

• Blocking and antibody incubation:

  • Block with 5% normal goat serum in PBS containing 0.1% Triton X-100 for 1 hour at room temperature

  • Dilute VPS28 antibody to 1:50-1:100 range in blocking solution

  • Extend primary antibody incubation to overnight at 4°C for maximal signal development

  • For co-labeling with neuronal markers, combine VPS28 antibody with anti-Tubb3 (β-III tubulin) antibodies, as this combination has been validated in mouse cortical neurons

• Signal amplification for low abundance detection:

  • If signal intensity is insufficient, implement tyramide signal amplification

  • Alternatively, use fluorophore-conjugated secondary antibodies with brighter fluorophores (Alexa Fluor 488 or 555)

  • Apply secondary antibodies at 1:500 dilution for 1-2 hours at room temperature

• Counterstaining and mounting:

  • Counterstain with DAPI (1:1000) to visualize nuclei

  • For long-term storage and to reduce photobleaching, mount slides with anti-fade mounting medium

• Imaging considerations:

  • Utilize confocal microscopy with appropriate optical sectioning to resolve endosomal structures

  • For co-localization studies with EVs markers, use super-resolution techniques when available

  • Compare VPS28 staining patterns in neuronal cell bodies versus neurites, as differential localization may occur

This protocol has been specifically tailored for neuronal applications based on successful VPS28 detection in cortical neurons and can be further optimized for specific neuronal subtypes or developmental stages.

What approaches can be used to study VPS28's role in VEGF-A trafficking?

To investigate VPS28's role in VEGF-A trafficking, which is crucial for its function in neurovascular communication , researchers should implement the following comprehensive approaches:

• Genetic manipulation of VPS28:

  • Generate VPS28 knockdown/knockout in neuronal cell lines or primary cultures using siRNA or CRISPR/Cas9

  • Create conditional knockout models for tissue-specific analysis (e.g., neuron-specific deletion)

  • Develop rescue experiments with wild-type or mutant VPS28 to identify critical domains

• VEGF-A detection methods:

  • Intracellular VEGF-A tracking:

    • Immunofluorescence co-localization of VPS28 (1:50-1:500 dilution) with VEGF-A antibodies

    • Live-cell imaging using fluorescently tagged VEGF-A constructs

  • Secreted VEGF-A quantification:

    • ELISA of conditioned media from control vs. VPS28-depleted cells

    • Western blot analysis of EVs isolated by ultracentrifugation to detect VEGF-A content

• EV isolation and characterization:

  • Isolate EVs from control and VPS28-depleted neuronal cells

  • Quantify EV numbers using nanoparticle tracking analysis (NTA)

  • Analyze VEGF-A content in isolated EVs using Western blot or ELISA

  • Perform proteomic analysis of EV cargo to identify additional VPS28-dependent trafficking pathways

• Functional assays:

  • Neuron-endothelial cell co-culture systems:

    • Seed neurons (control or VPS28-depleted) with endothelial cells

    • Measure endothelial cell migration, proliferation, and tube formation

    • Use VPS28 antibodies (1:50-1:500 for IF) to visualize protein in co-culture systems

  • Conditioned media transfer experiments:

    • Apply EVs isolated from control or VPS28-depleted neurons to endothelial cells

    • Assess activation of VEGF-R signaling pathways in recipient cells

    • Implement VEGF-A blocking antibodies to confirm specificity

• In vivo models:

  • Utilize zebrafish models where VPS28 has been knocked out

  • Examine vascular development, particularly central nervous system vascularization

  • Perform rescue experiments with neuronal-specific VPS28 expression

This multi-faceted approach combines molecular, cellular, and in vivo techniques to comprehensively characterize VPS28's specific role in regulating VEGF-A trafficking and neurovascular communication.

How can HRP-conjugated VPS28 antibodies be optimally used in immunohistochemistry applications?

For optimal use of HRP-conjugated VPS28 antibodies in immunohistochemistry applications, follow these specialized procedures:

• Tissue preparation and antigen retrieval:

  • Fix tissues appropriately (10% neutral buffered formalin recommended)

  • Perform heat-mediated antigen retrieval with citrate buffer pH 6, which is critical for VPS28 detection in paraffin sections

  • Allow slides to cool to room temperature before proceeding

• Endogenous enzyme blocking:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide in methanol for 10 minutes

  • For tissues with high endogenous peroxidase activity (e.g., liver, kidney), extend blocking time to 15-20 minutes

  • Wash thoroughly with PBS after blocking

• Protein blocking and antibody application:

  • Block non-specific binding with 5% normal serum in PBS for 1 hour at room temperature

  • For direct HRP-conjugated VPS28 antibodies, apply at optimized dilution (starting at 1:50 based on unconjugated antibody performance)

  • If signal amplification is needed, consider using biotinylated secondary antibodies followed by streptavidin-HRP

• Substrate development optimization:

  • Use 3,3′-diaminobenzidine (DAB) as a chromogen for standard brown visualization

  • Monitor color development under microscope to avoid overdevelopment

  • For multiplex IHC, consider alternative substrates that produce different colors

• Counterstaining considerations:

  • Counterstain lightly with hematoxylin to visualize tissue architecture without obscuring VPS28 signal

  • Blue hematoxylin provides good contrast with the brown DAB product

• Controls and validation:

  • Include isotype controls to assess non-specific binding

  • Use VPS28 knockout/knockdown tissues as negative controls

  • Include known positive tissues (kidney, brain) as positive controls

  • For doubled-check specificity, compare HRP-direct detection with indirect detection methods on serial sections

• Troubleshooting weak signals:

  • Increase antibody concentration

  • Extend incubation time (overnight at 4°C)

  • Optimize antigen retrieval conditions

  • Implement signal amplification systems (e.g., tyramide signal amplification)

These guidelines provide a comprehensive framework for optimizing HRP-conjugated VPS28 antibody performance in immunohistochemical applications across various tissue types.

What are the emerging research areas involving VPS28 beyond its established roles?

VPS28 research is expanding beyond its established role in the ESCRT-I complex to reveal novel functions in several exciting areas:

• Neurovascular development and brain function: Recent discoveries highlight VPS28's critical role in brain vasculature development through regulation of neuronal extracellular vesicle secretion and VEGF-A trafficking . This suggests potential implications in neurodevelopmental disorders and cerebrovascular diseases where neurovascular communication is disrupted.

• Extracellular vesicle biology: VPS28's involvement in EV secretion positions it as a key regulator of intercellular communication. Research is revealing how VPS28-dependent EV cargo selection may influence recipient cell function in diverse contexts beyond neurovascular interactions .

• Viral pathogenesis: As part of the ESCRT machinery, VPS28 may be exploited by viruses for budding and release. Investigating how different viruses interact with VPS28 could reveal novel antiviral targets.

• Neurodegenerative diseases: The enrichment of VPS28 in neurons suggests potential roles in protein homeostasis relevant to neurodegenerative conditions where protein aggregation and impaired vesicular trafficking are pathogenic mechanisms.

• Cancer biology: Dysregulation of ESCRT components affects receptor signaling and exosome production, potentially influencing tumor progression. VPS28's role in VEGF-A trafficking may have particular relevance for tumor angiogenesis.

These emerging research directions highlight VPS28's importance beyond basic cellular machinery, positioning it as a potential therapeutic target and biomarker for various pathological conditions where vesicular trafficking and intercellular communication are disrupted.

How might newer technologies enhance our understanding of VPS28 function?

Emerging technologies are poised to significantly advance our understanding of VPS28 function across multiple scales:

• Super-resolution microscopy: Techniques like STED, PALM, and STORM can resolve endosomal structures beyond the diffraction limit, allowing visualization of VPS28 localization within MVBs at unprecedented detail. Combined with VPS28 antibodies optimized for super-resolution applications, these approaches will reveal spatial organization previously hidden by conventional microscopy.

• Live-cell imaging advancements: New fluorescent protein tags with improved brightness and photostability, when combined with lattice light-sheet microscopy, will enable long-term tracking of VPS28 dynamics in living cells with minimal phototoxicity, providing insights into its temporal regulation.

• Proximity labeling proteomics: BioID or APEX2 fused to VPS28 would identify proximal interacting proteins in living cells, potentially uncovering novel associations beyond known ESCRT components. This approach is particularly valuable for identifying transient interactions within dynamic endosomal compartments.

• Single-vesicle analysis: Microfluidic approaches for isolating and analyzing individual extracellular vesicles will reveal how VPS28 influences the heterogeneity of EV populations, moving beyond bulk measurements to understand subpopulation-specific effects.

• Cryo-electron tomography: This technique can visualize the 3D ultrastructure of MVBs and associated ESCRT machinery at molecular resolution in a near-native state, potentially revealing how VPS28 contributes to membrane deformation during ILV formation.

• Single-cell multi-omics: Combining transcriptomics, proteomics, and metabolomics at the single-cell level will reveal how VPS28 expression correlates with broader cellular states and functions across diverse cell types, particularly in heterogeneous tissues like the brain where VPS28 shows enriched expression .

These technological advances will collectively provide multilayered insights into VPS28's functions, from molecular interactions to system-level impacts on intercellular communication and tissue development.

What are the potential therapeutic implications of targeting VPS28 in disease contexts?

Targeting VPS28 or its regulatory pathways presents several potential therapeutic opportunities based on its established biological functions:

• Neurovascular disorders: Given VPS28's critical role in regulating brain vasculature development through neuronal EV secretion and VEGF-A trafficking , modulating its activity could have therapeutic benefits in:

  • Stroke recovery, where neurovascular repair is critical

  • Vascular dementia, where cerebral blood flow is compromised

  • Neurodevelopmental disorders with vascular components

• Cancer therapy approaches:

  • Anti-angiogenic strategies: Inhibiting VPS28 in tumors could potentially reduce VEGF-A trafficking and secretion, complementing existing anti-VEGF therapies

  • EV-mediated cancer communication: Blocking VPS28-dependent release of tumor-derived EVs might reduce metastatic signaling and modulate the tumor microenvironment

• Viral infection interventions:

  • As ESCRT components are hijacked by viruses for budding, targeting VPS28 interactions could impair viral replication

  • Small molecule inhibitors of VPS28 binding interfaces could serve as broad-spectrum antivirals for ESCRT-dependent viruses

• Delivery system applications:

  • Engineering VPS28 expression could enhance therapeutic EV production

  • Manipulating VPS28 in producer cells might allow for improved loading of therapeutic cargo into EVs for targeted delivery

• Neurodegenerative disease approaches:

  • Given VPS28's enrichment in neurons , targeting its function could potentially modulate protein aggregation clearance mechanisms

  • Enhancing VPS28-dependent trafficking pathways might improve removal of pathogenic proteins