VPS36 Antibody, HRP conjugated

What is VPS36 and why is it important in cellular research?

VPS36 (Vacuolar Protein Sorting 36) is a component of the ESCRT-II complex (endosomal sorting complex required for transport II) that plays a critical role in 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 . Beyond its role in protein trafficking, the ESCRT-II complex containing VPS36 may also participate in transcription regulation through its interaction with ELL (elongation factor) . VPS36 is particularly important in research focused on membrane protein degradation pathways, ubiquitin-mediated sorting, and certain aspects of transcriptional regulation.

What are the primary applications for VPS36 antibodies in research?

VPS36 antibodies are valuable tools for several research applications:

• Western Blotting (WB) - Detection of VPS36 protein expression levels in cell or tissue lysates

• Immunofluorescence (IF) - Visualization of VPS36 subcellular localization

• Immunohistochemistry (IHC) - Examination of VPS36 expression in tissue sections

• Protein interaction studies - Investigation of VPS36 binding to other ESCRT components or ubiquitinated cargo

HRP-conjugated VPS36 antibodies are particularly useful for applications requiring enhanced sensitivity and chemiluminescent or colorimetric detection methods.

What is the cellular localization pattern of VPS36 when detected by antibodies?

VPS36 is primarily localized in the cytoplasm, as indicated in product documentation for commercially available antibodies . When visualized using immunocytochemistry or immunofluorescence, VPS36 typically displays a punctate cytoplasmic pattern that corresponds to endosomal structures, reflecting its role in the ESCRT machinery. The protein may also be detected in association with the plasma membrane during endocytosis of ubiquitinated membrane proteins . In certain contexts, a nuclear localization may be observed, consistent with its potential role in transcriptional regulation .

What are the key differences between unconjugated and HRP-conjugated VPS36 antibodies?

How do I select the most appropriate VPS36 antibody for my specific research application?

When selecting a VPS36 antibody, consider the following criteria:

• Target species: Ensure the antibody recognizes VPS36 in your species of interest. Available antibodies react with human, mouse, rat, and in some cases, additional species including cow, dog, and zebrafish .

• Application compatibility: Verify that the antibody has been validated for your intended application. Some VPS36 antibodies work well for WB but not for IF or IHC .

• Epitope location: Different antibodies target different regions of VPS36 (e.g., N-terminal, GLUE domain, or specific amino acid sequences like AA 87-105) . Choose an epitope region that is accessible in your experimental conditions.

• Conjugation needs: For direct detection without secondary antibodies, select HRP-conjugated VPS36 antibodies. For multiplexing or applications requiring amplification, unconjugated antibodies may be preferable .

• Validation data: Review the available validation data (Western blot images, immunofluorescence patterns) to ensure the antibody performs as expected in contexts similar to your planned experiments .

What are the optimal conditions for using HRP-conjugated VPS36 antibodies in Western blotting?

For optimal results with HRP-conjugated VPS36 antibodies in Western blotting:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease inhibitors

  • Use 20-50 μg of total protein per lane

  • Include phosphatase inhibitors if phosphorylation status is relevant

• Gel electrophoresis:

  • 12% SDS-PAGE is appropriate for resolving VPS36 (approximately 45 kDa)

  • Include positive controls (e.g., HEK293 or HeLa cell lysates)

• Transfer and blocking:

  • Transfer to PVDF membrane at 100V for 60-90 minutes

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Antibody incubation:

  • Dilute HRP-conjugated VPS36 antibody 1:1000-1:3000 in blocking buffer

  • Incubate overnight at 4°C for best results

  • Wash thoroughly with TBST (4-5 times, 5 minutes each)

• Detection:

  • Use enhanced chemiluminescence (ECL) substrate

  • Optimize exposure time based on signal intensity

  • For quantitative analysis, ensure signal is within linear range

How can I optimize immunofluorescence protocols when working with VPS36 antibodies?

While HRP-conjugated antibodies are not typically used for immunofluorescence, VPS36 detection by IF can be optimized as follows:

• Fixation method:

  • 4% paraformaldehyde (10-15 minutes at room temperature) preserves most epitopes

  • For certain applications, methanol fixation (-20°C, 10 minutes) may better preserve some epitopes

• Permeabilization:

  • 0.1-0.5% Triton X-100 in PBS for 5-10 minutes

  • Alternative: 0.1% saponin for milder permeabilization

• Antibody dilution and incubation:

  • Use VPS36 antibodies at 1:50-1:200 dilution as recommended

  • Incubate primary antibody overnight at 4°C

  • For co-localization studies, pair with markers for endosomes (EEA1, Rab5, Rab7) or MVBs (CD63)

• Controls and validation:

  • Include a peptide competition assay to confirm specificity

  • Consider siRNA knockdown of VPS36 as a negative control

  • Use appropriate blocking (5-10% normal serum from secondary antibody species)

What methods can be used to validate VPS36 antibody specificity in research applications?

Validating antibody specificity is crucial for reliable research. For VPS36 antibodies:

• Western blot validation:

  • Confirm band appears at expected molecular weight (~45 kDa for VPS36)

  • Test multiple cell lines with known VPS36 expression levels

  • Include negative controls (VPS36 knockout or knockdown samples)

  • Perform peptide competition assay with the immunizing peptide

• Immunoprecipitation followed by mass spectrometry:

  • Immunoprecipitate with the VPS36 antibody

  • Confirm the presence of VPS36 and known interacting partners (VPS22, VPS25) by mass spectrometry

• Genetic validation:

  • Compare antibody signal in wild-type versus VPS36 knockout/knockdown models

  • Use CRISPR-Cas9 edited cell lines with epitope tags on endogenous VPS36

• Cross-reactivity assessment:

  • Test antibody against recombinant proteins from closely related family members

  • Evaluate species cross-reactivity if working with non-human models

How can VPS36 antibodies be used to study ESCRT-II complex formation and function?

VPS36 antibodies can provide valuable insights into ESCRT-II biology:

• Co-immunoprecipitation studies:

  • Use VPS36 antibodies to immunoprecipitate the entire ESCRT-II complex

  • Detect associated components (VPS22, VPS25) by Western blotting

  • This approach can verify the assembly of the Y-shaped ESCRT-II structure in different cellular contexts

• Proximity ligation assays (PLA):

  • Combine VPS36 antibodies with antibodies against other ESCRT components

  • PLA can reveal in situ protein-protein interactions within intact cells

  • Quantify interaction signals under different experimental conditions

• Immunofluorescence co-localization:

  • Use VPS36 antibodies alongside markers for different endosomal compartments

  • Track the progressive movement of cargo through the ESCRT machinery

  • Analyze co-localization coefficients quantitatively

• Functional blocking studies:

  • Microinjection of VPS36 antibodies can potentially disrupt ESCRT-II function

  • Observe effects on MVB formation and cargo sorting in real-time

Importantly, VPS36 has been shown to interact directly with VPS22 and VPS25 in pull-down assays, supporting its role in ESCRT-II complex formation . These interactions are essential for the functional assembly of the ESCRT machinery.

What are the technical challenges in detecting VPS36 ubiquitin-binding activity using antibody-based methods?

Studying VPS36's ubiquitin-binding function presents several technical challenges:

• Epitope accessibility issues:

  • Antibodies targeting the GLUE domain might interfere with ubiquitin binding

  • Consider using antibodies against distant epitopes when studying ubiquitin interactions

• Detecting transient interactions:

  • VPS36-ubiquitin binding is often transient and may require crosslinking

  • Use membrane-permeable crosslinkers (e.g., DSP) prior to cell lysis

  • Co-immunoprecipitation with ubiquitin antibodies can help capture these interactions

• Species-specific differences:

  • Unlike yeast, Arabidopsis VPS36 shows ubiquitin binding even without the GLUE domain

  • Human VPS36 has different ubiquitin-binding mechanisms than yeast counterparts

  • Carefully consider species differences when designing experiments

• Experimental validation:

  • Use ubiquitin agarose pull-down assays to confirm binding capability

  • Compare full-length VPS36 with GLUE domain-deleted constructs

  • Employ ubiquitin mutants to map specific binding interfaces

Research has shown that Arabidopsis VPS36 exhibits ubiquitin-binding through its GLUE domain, but interestingly, even VPS36ΔGLUE can interact with ubiquitin, suggesting additional binding motifs not present in yeast homologs .

What approaches can be used to study VPS36 phosphoinositide binding using antibody-based detection methods?

VPS36 has been reported to bind phosphoinositides such as PtdIns(3,4,5)P3 . To study this interaction:

• Protein-lipid overlay assays:

  • Use recombinant VPS36 or immunoprecipitated VPS36 with phosphoinositide arrays

  • Detect bound VPS36 with specific antibodies

  • Compare binding profiles of full-length protein versus isolated domains

• Liposome flotation assays:

  • Prepare liposomes containing different phosphoinositides

  • Incubate with cell lysates or recombinant VPS36

  • Fractionate by centrifugation and detect VPS36 by immunoblotting

• Cellular localization studies:

  • Treat cells with PI3K inhibitors to deplete PtdIns(3,4,5)P3

  • Monitor changes in VPS36 localization by immunofluorescence

  • Co-stain with markers for phosphoinositide-rich endosomal compartments

• Domain-specific mutants:

  • Generate VPS36 constructs with mutations in the GLUE domain

  • Compare phosphoinositide binding between wild-type and mutant proteins

  • Use antibodies against tags (if using tagged constructs) or against VPS36 directly

How can I address high background issues when using HRP-conjugated VPS36 antibodies in Western blots?

High background with HRP-conjugated antibodies can be addressed through these strategies:

• Optimization of antibody concentration:

  • Titrate the antibody (try 1:1000, 1:2000, 1:5000 dilutions)

  • Remember that HRP-conjugated antibodies often require higher dilutions than unconjugated antibodies

• Blocking optimization:

  • Test different blocking agents (5% milk, 3-5% BSA, commercial blockers)

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

  • Add 0.1-0.3% Tween-20 to blocking buffer to reduce hydrophobic interactions

• Washing improvements:

  • Increase washing frequency (5-6 washes instead of 3)

  • Extend wash duration (10 minutes per wash)

  • Use fresh TBST for each wash

• Sample preparation:

  • Ensure complete protein denaturation (heat samples at 95°C for 5 minutes)

  • Pre-clear lysates by centrifugation at high speed

  • Filter lysates through 0.45 μm filters to remove particulates

• Membrane handling:

  • Never allow membrane to dry during the protocol

  • Optimize transfer conditions to prevent protein over-transfer

  • Consider low-fluorescence PVDF membranes for lower background

What are the best approaches for multiplexing VPS36 detection with other ESCRT components?

For comprehensive analysis of the ESCRT machinery:

• Sequential immunoblotting:

  • Use HRP-conjugated VPS36 antibody first

  • Strip the membrane using commercial stripping buffer (validate complete stripping)

  • Re-probe with antibodies against other ESCRT components

  • Use different visualization methods (e.g., chemiluminescence vs. fluorescence) to distinguish signals

• Multi-color immunofluorescence:

  • Select antibodies raised in different host species (e.g., rabbit anti-VPS36, mouse anti-VPS25)

  • Use species-specific secondary antibodies with distinct fluorophores

  • Include appropriate controls for antibody cross-reactivity

  • Apply spectral unmixing if fluorophore emission spectra overlap

• Proximity ligation assay (PLA):

  • Combine VPS36 antibodies with antibodies against interaction partners

  • PLA generates punctate fluorescent signals only where proteins are in close proximity

  • Quantify interaction signals to measure complex formation

• Mass cytometry (CyTOF):

  • Label antibodies with different metal isotopes

  • Simultaneously detect multiple ESCRT components without fluorescence overlap limitations

  • Provides single-cell resolution for heterogeneous populations

How do I interpret discrepancies in VPS36 detection between different antibodies or detection methods?

When facing inconsistent results with VPS36 antibodies:

• Epitope accessibility differences:

  • Different antibodies target distinct regions (N-terminal, GLUE domain, aa 87-105, etc.)

  • Some epitopes may be masked in protein complexes or certain conformational states

  • Compare results from antibodies targeting different regions of VPS36

• Post-translational modifications:

  • Phosphorylation or ubiquitination may affect epitope recognition

  • Some antibodies may preferentially recognize modified/unmodified forms

  • Use phosphatase treatment or deubiquitinating enzymes to test this possibility

• Isoform specificity:

  • Check if antibodies recognize all known VPS36 isoforms

  • Verify which splice variants are expressed in your experimental system

  • Design PCR primers to confirm isoform expression at the mRNA level

• Methodological differences:

  • Fixation methods can dramatically affect epitope preservation (formaldehyde vs. methanol)

  • Denaturation conditions in Western blotting influence protein conformation

  • Native conditions (co-IP) may preserve interactions that mask certain epitopes

• Validation approach:

  • Use genetic approaches (siRNA, CRISPR) to confirm specificity

  • Compare with tagged VPS36 constructs detected via the tag

  • Consider absolute quantification methods (mass spectrometry) as an orthogonal approach

How might advances in antibody technology improve our understanding of VPS36 function?

Emerging antibody technologies could enhance VPS36 research:

• Conformation-specific antibodies:

  • Develop antibodies that specifically recognize VPS36 in ESCRT-II complex

  • Generate antibodies detecting ubiquitin-bound versus unbound states

  • Create phospho-specific antibodies if regulatory phosphorylation sites are identified

• Intrabodies and nanobodies:

  • Express VPS36-targeting antibody fragments within living cells

  • Monitor real-time dynamics of VPS36 in intact cellular environments

  • Use for acute disruption of specific VPS36 interactions

• BiTE (Bi-specific T-cell Engager) approach for protein interaction studies:

  • Develop bi-specific antibodies targeting VPS36 and potential interacting partners

  • Use to validate and characterize novel protein-protein interactions

  • Apply in various cellular contexts to map interaction networks

• Degradation-inducing antibodies:

  • Create VPS36-targeting PROTACs or dTAGs

  • Enable rapid, inducible degradation of VPS36 to study acute loss effects

  • Compare with genetic knockout to distinguish between acute and adaptive responses

What methodological advances could improve quantitative analysis of VPS36 in MVB biogenesis?

Advanced methods for quantitative VPS36 studies include:

• Super-resolution microscopy approaches:

  • Apply STORM or PALM imaging with VPS36 antibodies

  • Achieve nanometer-scale resolution of VPS36 localization

  • Quantify spatial relationships between VPS36 and other ESCRT components

• Live-cell imaging with split fluorescent proteins:

  • Engineer cells with VPS36 fused to one half of a split fluorescent protein

  • Fuse interaction partners with complementary half

  • Monitor complex assembly in real-time when fluorescence complementation occurs

• Quantitative mass spectrometry:

  • Use stable isotope labeling (SILAC) combined with immunoprecipitation

  • Identify and quantify the stoichiometry of VPS36 interactions

  • Compare interaction profiles under different cellular conditions

• Correlative light and electron microscopy (CLEM):

  • Localize VPS36 using fluorescently-labeled antibodies

  • Correlate with ultrastructural features by electron microscopy

  • Precisely map VPS36 to specific regions of forming MVBs

How can VPS36 antibodies contribute to our understanding of disease mechanisms?

VPS36 antibodies can provide insights into disease processes:

• Neurodegenerative disease research:

  • ESCRT dysfunction is implicated in neurodegenerative disorders

  • Use VPS36 antibodies to examine protein trafficking in disease models

  • Investigate co-localization with disease-associated proteins (α-synuclein, tau, etc.)

• Cancer biology applications:

  • Analyze VPS36 expression in tumor tissue microarrays

  • Correlate with cancer progression and treatment response

  • Investigate relationship to receptor tyrosine kinase downregulation

• Viral pathogenesis studies:

  • Many viruses hijack the ESCRT machinery for budding

  • Use VPS36 antibodies to track ESCRT recruitment to viral assembly sites

  • Develop assays for compounds that disrupt these interactions

• Autophagy and cellular stress responses:

  • Examine VPS36 redistribution during autophagy induction

  • Monitor changes in complex formation during cellular stress

  • Develop biomarkers for autophagic dysfunction based on VPS36 status