VPS36 Antibody, FITC conjugated

What is VPS36 and why is it significant for cellular research?

VPS36 (Vacuolar Protein-Sorting-associated protein 36) is a critical component of the endosomal sorting complex required for transport II (ESCRT-II), which is essential for multivesicular body (MVB) formation and sorting of endosomal cargo proteins into MVBs . The MVB pathway mediates delivery of transmembrane proteins into the lysosome lumen for degradation. VPS36 is significant because:

• It forms a complex with VPS25, VPS22/SNF8 to create the functional ESCRT-II complex

• It possesses ubiquitin-binding activity through its GLUE (GRAM-like ubiquitin-binding in EAP45) domain

• It bridges the interaction between different ESCRT complexes, making it crucial for the MVB pathway

• It may also play a role in transcription regulation through its interaction with ELL (elongation factor)

Recent studies demonstrate that VPS36 is critical for cellular processes ranging from embryonic development to membrane protein degradation , making it an important target for cellular biology research.

The specificity of VPS36 antibodies is confirmed through multiple validation approaches:

• Western blotting validation: Antibodies are tested against cell or tissue lysates to verify they detect a single band at the expected molecular weight of approximately 43-44 kDa . Some antibodies may show the verification of specificity through blocking peptide controls, where pre-incubation with the immunizing peptide abolishes the signal .

• Immunofluorescence co-localization: Validation by co-staining with known markers of late endosomes and verification that the pattern matches the expected subcellular localization (cytoplasm, endosome, late endosome, membrane, and nucleus) .

• Cross-reactivity testing: Testing against multiple species to determine reactivity profile. Most VPS36 antibodies are validated against human, mouse, and rat samples, with some showing broader cross-reactivity to other mammals .

• Knockout/knockdown validation: Some advanced validation methods include using VPS36-knockdown or knockout cells to confirm antibody specificity.

When selecting a VPS36 antibody for research, verification of species reactivity and application-specific validation are essential for reliable results.

What are the optimal protocols for using FITC-conjugated VPS36 antibodies in immunofluorescence studies?

For optimal immunofluorescence results with FITC-conjugated VPS36 antibodies:

• Sample preparation:

  • Fix cells using 4% paraformaldehyde for 15-20 minutes at room temperature

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

  • Block with 1-5% bovine serum albumin (BSA) or normal serum for 30-60 minutes

• Antibody dilution:

  • Use a 1:50-1:200 dilution of the FITC-conjugated VPS36 antibody

  • Dilute in blocking buffer containing 0.1% Triton X-100

• Incubation conditions:

  • Incubate overnight at 4°C in a humidified chamber

  • Protect from light to prevent photobleaching of the FITC fluorophore

• Washing steps:

  • Wash 3-5 times with PBS containing 0.1% Tween-20

  • Each wash should be 5-10 minutes with gentle agitation

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes

  • Mount using an anti-fade mounting medium to prevent photobleaching

For co-localization studies, researchers can co-stain with other subcellular markers like endosomal markers (EEA1 for early endosomes, RAB7 for late endosomes) to confirm the specificity of VPS36 localization to late endosomes as reported in the literature .

How do researchers distinguish between normal and pathological VPS36 expression patterns?

Distinguishing normal from pathological VPS36 expression patterns requires:

• Baseline establishment: Researchers should first establish baseline expression patterns in appropriate control tissues or cell lines. VPS36 typically shows punctate cytoplasmic staining with enrichment in endosomal structures .

• Quantitative analysis:

  • Measure fluorescence intensity using software like ImageJ

  • Quantify the number and size of VPS36-positive puncta

  • Compare expression levels between normal and pathological samples using Western blotting

• Co-localization analysis:

  • In normal cells, VPS36 colocalizes with ubiquitinated proteins on late endosomes

  • Aberrant localization may indicate pathological conditions

• Functional assessment:

  • Normal VPS36 function can be assessed by examining ESCRT-dependent processes

  • Disruption of MVB formation or accumulation of ubiquitinated proteins indicates dysfunction

  • Overexpression of dominant-negative VPS4 (E228Q mutant) can be used as a positive control for ESCRT dysfunction, as it leads to accumulation of ubiquitinated proteins and enlarged vacuole-like structures

Comparing these parameters between normal and pathological samples allows researchers to identify alterations in VPS36 expression or function that may contribute to disease mechanisms.

What are the functional implications of VPS36's ubiquitin-binding activity in experimental designs?

VPS36's ubiquitin-binding activity has significant functional implications that researchers should consider in experimental designs:

• ESCRT pathway studies: VPS36 binds ubiquitinated membrane proteins through its GLUE domain, making it a critical link in the ESCRT-mediated degradation pathway . Experiments targeting this interaction can reveal mechanisms of membrane protein degradation.

• Ubiquitin binding assays: The VPS36 GLUE domain has been shown to bind ubiquitin in pull-down assays, and interestingly, even VPS36ΔGLUE (with the GLUE domain deleted) retains some ubiquitin affinity, suggesting additional binding sites . This can be leveraged in experimental designs to:

  • Use ubiquitin agarose as bait for pull-down assays

  • Compare binding affinities of different VPS36 domains

  • Test effects of mutations on ubiquitin binding

• Cargo recognition studies: Experiments can be designed to identify specific ubiquitinated cargo recognized by VPS36, potentially revealing novel substrates of the ESCRT pathway.

• Structural studies: Understanding the structural basis of VPS36-ubiquitin interaction can guide the design of inhibitors or enhancers of this interaction.

• Inter-species comparisons: Unlike yeast VPS36 which interacts with ubiquitin through an NZF motif, plant and animal VPS36 proteins lack this motif . Comparative studies can reveal evolutionary adaptations in ubiquitin recognition.

Researchers can use FITC-conjugated VPS36 antibodies in combination with antibodies against ubiquitinated proteins (such as FK2 antibody) to visualize colocalization and track changes in response to perturbations in the ubiquitin system .

How does VPS36 function in the context of the ESCRT-II complex, and what experimental approaches best reveal these interactions?

VPS36 functions within the ESCRT-II complex through specific interactions that can be studied using the following experimental approaches:

• Complex composition analysis:

  • The ESCRT-II complex consists of VPS36, VPS25 (two copies), and SNF8/VPS22

  • Co-immunoprecipitation experiments confirm these interactions

  • VPS36 interaction with VPS22 and VPS25 can be demonstrated by GST pull-down assays

• Sequential recruitment studies:

  • VPS36 connects ESCRT-I (through interaction with TSG101/VPS28) to ESCRT-III (through CHMP6)

  • These connections can be studied using fluorescently labeled proteins and live-cell imaging

  • Proximity ligation assays can detect protein-protein interactions in situ

• Functional domain mapping:

  • VPS36 contains a GLUE domain for ubiquitin binding and a VPS36 domain for interacting with VPS22 and VPS25

  • Truncation mutants can reveal the function of specific domains

  • For example, GST-VPS36ΔGLUE can be used to test interactions independent of the GLUE domain

• Visualization of complex assembly:

  • FITC-conjugated VPS36 antibodies allow direct visualization of VPS36 localization

  • Co-staining with antibodies against other ESCRT components reveals their spatial relationships

  • 3D reconstruction of images can show close localization, as demonstrated for AKTIP and TSG101 at the midbody

• Viral interference studies:

  • Some viruses, like foot-and-mouth disease virus (FMDV), target ESCRT components

  • FMDV 3C protease degrades Vps28, and similar mechanisms may affect other ESCRT components

  • These viral interactions provide natural experiments to understand ESCRT function

The ESCRT-II complex bridges ESCRT-I and ESCRT-III, making VPS36 a key player in the sequential recruitment of ESCRT machinery to endosomal membranes for MVB formation .

What are the most effective positive and negative controls for VPS36 antibody validation?

Effective controls for VPS36 antibody validation include:

Positive Controls:

• Cell lines with confirmed VPS36 expression:

  • Human cell lines: HeLa, 293T cells have been documented to express VPS36

  • Mouse cell lines: NIH/3T3 has been verified for IF applications

• Tissue samples:

  • Rat liver and kidney have been validated for Western blotting

  • Brain tissue has been validated for immunohistochemistry

• Recombinant VPS36 protein:

  • Full-length (positions M1-S386) or domain-specific recombinant proteins

  • Synthetic peptides corresponding to specific regions (e.g., aa 87-105, 250-310)

• Overexpression systems:

  • Cells transfected with VPS36 expression constructs

  • Tagged VPS36 (e.g., GST-VPS36) that can be detected with tag-specific antibodies

Negative Controls:

• Blocking peptide competition:

  • Pre-incubation of the antibody with the immunizing peptide should abolish specific staining

  • This control confirms the antibody is binding to its intended epitope

• VPS36 knockdown/knockout:

  • siRNA-mediated knockdown of VPS36

  • CRISPR/Cas9-generated VPS36 knockout cell lines

• Isotype controls:

  • Rabbit IgG isotype control at the same concentration as the VPS36 antibody

  • For FITC-conjugated antibodies, FITC-conjugated isotype controls are essential

• Secondary antibody-only controls:

  • For non-conjugated primary antibodies, omitting the primary antibody while retaining the secondary antibody

These controls ensure that the observed signal is specific to VPS36 and not due to non-specific binding or autofluorescence.

How does VPS36 interact with other components of the endosomal sorting machinery beyond the ESCRT-II complex?

VPS36 interacts with multiple proteins beyond the ESCRT-II complex, forming a network essential for endosomal sorting:

• Interaction with ESCRT-I components:

  • VPS36 interacts with VPS28, a component of ESCRT-I

  • Co-immunoprecipitation and GST pull-down assays show that VPS36 does not interact with VPS25, VPS22 but confirms its interaction with VPS28

  • VPS36, VPS28, and TSG101 (another ESCRT-I component) can be captured together in pull-down assays, suggesting they form a complex

• Connection to ESCRT-III recruitment:

  • VPS36 interacts with CHMP6, an ESCRT-III component

  • This interaction facilitates the sequential recruitment of ESCRT machinery

• Regulatory protein interactions:

  • VPS36 interacts with RILPL1 (via the C-terminal domain), which recruits ESCRT-II to endosome membranes

  • Interaction with ECPAS has been reported, though its functional significance requires further investigation

• Relationship with AKTIP:

  • AKTIP interacts with VPS28 and TSG101, and these proteins colocalize at the midbody

  • 99% of midbodies positive for AKTIP are also positive for TSG101, indicating their coordinated function

• VPS4-dependent processes:

  • The AAA-ATPase VPS4 is responsible for disassembling ESCRT-III filaments

  • Overexpression of dominant-negative VPS4 (E228Q) delays degradation of ubiquitinated proteins, demonstrating the functional connection between VPS36-containing complexes and VPS4 activity

These interactions can be studied using various approaches, including co-immunoprecipitation, proximity ligation assays, and fluorescence microscopy with FITC-conjugated VPS36 antibodies in combination with antibodies against interacting partners.

What are the key troubleshooting considerations when working with FITC-conjugated VPS36 antibodies?

When working with FITC-conjugated VPS36 antibodies, researchers should consider these key troubleshooting points:

• Signal intensity issues:

  • Problem: Weak fluorescence signal

  • Solutions:

    • Optimize antibody concentration (try 1:50 dilution for stronger signal)

    • Extend incubation time to overnight at 4°C

    • Use antigen retrieval techniques for fixed tissues

    • Check sample preparation methods; overfixation can mask epitopes

• Photobleaching concerns:

  • Problem: FITC signal fades during imaging

  • Solutions:

    • Use anti-fade mounting medium

    • Minimize exposure to light during sample preparation

    • Use lower intensity excitation light and shorter exposure times

    • Consider capturing images from unexposed areas of the slide first

• Background and specificity issues:

  • Problem: High background or non-specific staining

  • Solutions:

    • Increase blocking time and concentration (use 5% BSA)

    • Add 0.1-0.3% Triton X-100 to antibody dilution buffer

    • Include additional washing steps

    • Validate observed patterns with non-conjugated VPS36 antibodies

    • Compare staining pattern with expected subcellular localization (cytoplasm, endosome, late endosome, membrane, nucleus)

• Molecular weight discrepancies:

  • Problem: Detected band size does not match expected 43-44 kDa

  • Solutions:

    • Note that the observed molecular weight (43 kDa) may not match the calculated MW (36 kDa)

    • This discrepancy can result from post-translational modifications

    • Use positive controls with known VPS36 expression

    • Consider whether splice variants might be present in your sample

• Storage and stability concerns:

  • Problem: Antibody performance deteriorates over time

  • Solutions:

    • Store at -20°C for long-term storage

    • Avoid repeated freeze-thaw cycles by preparing small aliquots

    • For reconstituted antibodies, store at 4°C for short-term use (up to one month)

    • Protect from light to preserve FITC fluorescence

• Cross-reactivity concerns:

  • Problem: Uncertainty about species cross-reactivity

  • Solutions:

    • Verify the antibody's validated species reactivity before use

    • Most VPS36 antibodies react with human, mouse, and rat samples

    • For non-validated species, perform preliminary validation experiments

Following these troubleshooting guidelines will help ensure reliable and reproducible results when working with FITC-conjugated VPS36 antibodies in research applications.

How can researchers leverage VPS36 antibodies to study interactions between viruses and the ESCRT machinery?

Researchers can use VPS36 antibodies to investigate virus-ESCRT interactions through the following approaches:

• Viral evasion mechanisms study:

  • Foot-and-mouth disease virus (FMDV) has been shown to downregulate Vps28, an ESCRT-I component that interacts with VPS36

  • FITC-conjugated VPS36 antibodies can be used to track changes in VPS36 localization during viral infection

  • Western blot analysis can determine whether viruses also target VPS36 for degradation or relocalization

• ESCRT-dependent viral budding:

  • Many enveloped viruses hijack the ESCRT machinery for budding

  • Co-immunostaining with viral proteins and VPS36 can reveal recruitment to budding sites

  • Changes in VPS36 distribution during infection can indicate viral manipulation of ESCRT complexes

• Viral protein-ESCRT interaction:

  • FMDV 3C protease mediates Vps28 degradation through autophagy

  • Similar mechanisms may target VPS36 or other ESCRT components

  • Co-immunoprecipitation using VPS36 antibodies can identify viral proteins that interact with ESCRT-II

• Functional antiviral roles:

  • Overexpression of Vps28 decreases FMDV replication

  • Similar experiments with VPS36 can reveal potential antiviral functions

  • FITC-conjugated VPS36 antibodies can be used in flow cytometry to correlate VPS36 expression levels with viral replication in cell populations

• Viral replication tracking:

  • Monitor changes in VPS36 localization and expression during different stages of viral infection

  • Time-course experiments using immunofluorescence can reveal dynamic changes in ESCRT function

• ESCRT perturbation effects on viral replication:

  • Use dominant-negative mutants of ESCRT components to study their impact on viral replication

  • Compare viral replication in cells with normal versus disrupted ESCRT function

These approaches can provide insights into both how viruses manipulate the ESCRT machinery and how the ESCRT system might function in antiviral defense mechanisms.

What advanced microscopy techniques are most effective when working with FITC-conjugated VPS36 antibodies?

Advanced microscopy techniques can significantly enhance research with FITC-conjugated VPS36 antibodies:

• Confocal microscopy:

  • Provides high-resolution imaging of VPS36 localization in cellular compartments

  • Optical sectioning eliminates out-of-focus fluorescence

  • Z-stack acquisition allows 3D reconstruction of VPS36 distribution

  • Example application: 3D reconstruction has shown that AKTIP and TSG101 closely localize at the midbody

• Super-resolution microscopy:

  • Structured Illumination Microscopy (SIM) provides resolution beyond the diffraction limit

  • Stimulated Emission Depletion (STED) microscopy can reveal fine details of VPS36-positive structures

  • Single-Molecule Localization Microscopy (PALM/STORM) can map individual VPS36 molecules with nanometer precision

  • These techniques can resolve individual endosomes and MVBs that may appear as single puncta in conventional microscopy

• Live-cell imaging:

  • For transfected fluorescent protein-tagged VPS36

  • Enables tracking of dynamics in real-time

  • Photobleaching techniques (FRAP/FLIP) can measure VPS36 mobility and turnover

  • Note: FITC-conjugated antibodies are typically used in fixed cells, but complementary live imaging with tagged proteins can provide dynamic information

• Correlative Light and Electron Microscopy (CLEM):

  • Combines fluorescence localization of VPS36 with ultrastructural context

  • Can definitively identify VPS36-positive structures as MVBs, endosomes, or other compartments

  • Immunogold labeling for EM can confirm VPS36 localization at the ultrastructural level

• Automated high-content imaging:

  • Enables quantitative analysis of VPS36 distribution across large cell populations

  • Can detect subtle changes in localization or expression levels

  • Particularly useful for screening experiments or analyzing heterogeneous responses

• Proximity Ligation Assay (PLA):

  • Detects protein-protein interactions in situ with high sensitivity

  • Can confirm VPS36 interactions with other ESCRT components

  • Example application: Could verify the close association of VPS36 with VPS28 or TSG101 seen in biochemical assays