VPS36 Antibody, Biotin conjugated

Advanced Experimental Considerations

• How can researchers optimize immunofluorescence protocols for co-localization studies involving biotin-conjugated VPS36 antibodies?

  Co-localization studies with biotin-conjugated VPS36 antibodies require careful optimization to achieve accurate results:

  1. Sequential staining protocol:

     • Fix cells with 4% paraformaldehyde (10 minutes, room temperature)

     • Permeabilize with 0.1% Triton X-100 (5 minutes)

     • Block with 10% normal goat serum (30 minutes)

     • Incubate with primary antibodies for co-localization marker first

     • Apply biotin-conjugated VPS36 antibody at 1:100-1:200 dilution (overnight at 4°C)

     • Detect with streptavidin-conjugated fluorophore (AlexaFluor 594 recommended )

     • Counterstain with DAPI and mount

  2. Critical controls:

     • Single staining controls to assess bleed-through

     • Secondary-only controls to evaluate background

     • Blocking of endogenous biotin using avidin/biotin blocking kit

  3. Advanced considerations:

     • When studying endosomal compartments, use VPS36 (1:100) with early endosome marker EEA1 or late endosome marker Rab7

     • For ESCRT complex studies, co-stain with VPS25 (1:150) and VPS22 (1:200)

     • For ubiquitinated protein tracking, combine with ubiquitin antibodies (1:250)

  Successful co-localization experiments have demonstrated VPS36 association with ubiquitinated proteins on late endosomes and its recruitment to endosome membranes during vesicle formation .

• What approaches are recommended for studying the interaction between VPS36 and other ESCRT-II components?

  Investigating VPS36 interactions with other ESCRT-II components requires multiple complementary techniques:

  1. In vitro protein pull-down assays: Recombinant proteins (GST-VPS36, 6xHis-VPS22, and 6xHis-VPS25-6xHis) can be used to demonstrate direct interactions. Glutathione sepharose beads bound to GST-VPS36 efficiently pull down 6xHis-VPS22 and 6xHis-VPS25-6xHis, confirming their association in the ESCRT-II complex .

  2. Co-immunoprecipitation: For endogenous interactions, use biotin-conjugated VPS36 antibody (1:200) with streptavidin beads, followed by western blot analysis for VPS22 and VPS25.

  3. Proximity ligation assay (PLA): This technique can detect protein-protein interactions in situ with high sensitivity, providing spatial information about interaction sites.

  4. Yeast two-hybrid screening: This approach has successfully identified interacting partners of VPS36, including confirmation of interactions with VPS22, VPS25, and novel interactions with proteins like Arl4A .

  5. Structure-function analysis: Site-directed mutagenesis of key residues in the VPS36 domain (like the conserved 352LAKER356 region) can disrupt specific interactions, as demonstrated in the Arl4A-VPS36 interaction .

  These approaches have revealed that VPS36 forms a Y-shaped structure with VPS22 and two VPS25 molecules, which is critical for ESCRT-II function .

• How should researchers address the challenges of specificity when using biotin-conjugated antibodies in tissues with high endogenous biotin?

  Endogenous biotin presents significant challenges when using biotin-conjugated antibodies, particularly in tissues with high biotin content (liver, kidney, brain). Researchers should implement these methodological solutions:

  1. Endogenous biotin blocking:

     • Apply avidin/biotin blocking kit before antibody incubation

     • Alternative protocol: pre-incubate sections with 0.1% avidin (15 minutes), wash, then 0.01% biotin (15 minutes)

  2. Tissue-specific considerations:

     • For rat liver and kidney samples (verified for VPS36 antibody ), increase blocking time to 45 minutes

     • Include 0.1% Tween-20 in wash buffers to reduce non-specific binding

  3. Detection strategy modifications:

     • Use streptavidin polymers rather than monomeric streptavidin for improved signal-to-noise ratio

     • Consider tyramide signal amplification (TSA) for specific signal enhancement

     • In multiplex IHC, apply the biotin-conjugated VPS36 antibody in the final detection step

  4. Validation controls:

     • Include tissue sections treated with streptavidin-conjugate alone

     • Compare results with unconjugated VPS36 antibody using conventional detection

     • Process biotin-free control tissues in parallel

  These strategies are essential particularly when using biotin-conjugated VPS36 antibodies in metabolically active tissues where endogenous biotin might otherwise produce misleading results.

Research Applications and Troubleshooting

• What is the role of VPS36 in the ESCRT pathway, and how can researchers effectively study its function in protein trafficking?

  VPS36 serves as a critical linker in the ESCRT pathway through multiple domain-specific functions:

  1. Functional domains and mechanisms:

     • The GLUE domain binds both phosphoinositides and ubiquitin

     • The VPS36 domain mediates interaction with other ESCRT-II components

     • Through these interactions, VPS36 facilitates cargo recognition, sorting, and ESCRT complex assembly

  2. Methodological approaches for functional studies:

     • RNA interference: siRNA targeting VPS36 (demonstrated in Arabidopsis studies) shows defects in endosomal sorting and vacuolar biogenesis

     • Dominant-negative mutants: Expression of truncated VPS36 constructs disrupts normal ESCRT function

     • Cargo trafficking assays: Using fluorescently-labeled EGFR to track degradation kinetics in the presence/absence of VPS36

     • Proximity labeling: BirA fusion proteins to identify proteins in the microenvironment of the ESCRT complex

  3. Visualization techniques:

     • Live cell imaging with VPS36-GFP fusion proteins to track dynamic ESCRT assembly

     • APEX2 fusion proteins for electron microscopy visualization of VPS36 localization at ultrastructural level

  Recent research has revealed VPS36's role beyond traditional ESCRT functions, including interactions with Arl4A to regulate EGFR degradation and potential roles in transcriptional regulation through interaction with ELL.

• How do researchers interpret variable VPS36 banding patterns in Western blot analysis?

  Variability in VPS36 Western blot banding patterns presents a significant analytical challenge requiring systematic interpretation:

  1. Expected vs. observed band patterns:

     • Predicted molecular weight: 44 kDa (based on amino acid sequence)

     • Commonly observed: Primary band at 43 kDa with occasional secondary band at 65 kDa

  2. Sources of variation and interpretation:

     Banding Pattern	Possible Explanation	Verification Method
     Single band at 43-44 kDa	Canonical VPS36	Peptide competition
     Double bands (43-44 kDa + 65 kDa)	Post-translational modifications or alternative splicing	Phosphatase treatment, isoform-specific antibodies
     Multiple bands	Non-specific binding or protein degradation	Fresh sample preparation, protease inhibitors
     Shifted bands in different tissues	Tissue-specific post-translational modifications	Compare multiple antibodies targeting different epitopes

  3. Optimization strategies:

     • Adjust antibody dilution (1:500-1:2000 recommended for biotin-conjugated VPS36 antibody)

     • Optimize blocking conditions (5% non-fat milk in TBS recommended)

     • Consider gradient gels (5-20% SDS-PAGE) for better resolution

     • For biotin-conjugated antibodies, use streptavidin-HRP at 1:5000-1:10000 dilution

  4. Validation approaches:

     • Compare multiple VPS36 antibodies targeting different epitopes

     • Include lysates from VPS36-depleted cells as negative controls

     • Use recombinant VPS36 protein as positive control

  Researchers have documented consistent detection at 43-44 kDa across multiple cell lines including HL-60, THP-1, MOLT-4, and U937, as well as in rat and mouse brain tissue lysates .

• What are the considerations for using biotin-conjugated VPS36 antibodies in proximity-dependent biotinylation (BioID) experiments?

  When incorporating biotin-conjugated VPS36 antibodies in proximity-dependent biotinylation studies, researchers must address several methodological considerations:

  1. Experimental design challenges:

     • Biotin-conjugated antibodies may interfere with BioID analysis through background biotinylation

     • Careful controls must distinguish antibody-derived biotin from proximity-dependent biotinylation

  2. Recommended protocol modifications:

     • Use split BioID systems where BirA is fused to a candidate interacting partner of VPS36

     • Employ two-step labeling: first detecting VPS36 with biotin-conjugated antibodies, then performing BioID with a different fusion protein

     • Implement differential elution strategies to separate antibody-biotinylated proteins from BioID-labeled proteins

  3. Alternative approaches:

     • Replace BioID with APEX2-based proximity labeling when using biotin-conjugated antibodies

     • Use epitope-tagged VPS36 constructs (HA, FLAG) with corresponding unconjugated antibodies

     • Consider TurboID for faster labeling kinetics with shorter biotin exposure times

  4. Advanced analysis strategies:

     • Quantitative proteomics with SILAC or TMT labeling to distinguish true interactions

     • Computational filtering to remove common background proteins

     • Spatial mapping using site-specific biotinylation patterns

  The proximity-dependent biotinylation approach has successfully identified >500 host proteins associated with viral replication complexes , demonstrating its value for studying protein interaction networks when properly controlled.

• How can researchers effectively use biotin-conjugated VPS36 antibodies to investigate the relationship between the ESCRT pathway and human disease models?

  Biotin-conjugated VPS36 antibodies offer powerful tools for investigating ESCRT pathway dysregulation in disease models:

  1. Neurodegenerative diseases:

     • Measure VPS36 expression and localization in Alzheimer's or Parkinson's disease models

     • Co-localization studies with disease-associated proteins (Tau, α-synuclein) using dual immunofluorescence

     • Quantitative assessment of endolysosomal pathway alterations using biotin-conjugated VPS36 (1:100) with endosomal markers

  2. Cancer research applications:

     • Tissue microarray analysis of VPS36 expression across cancer types

     • Correlation of ESCRT component expression with patient outcomes

     • Investigation of EGFR trafficking defects in cancer cells using VPS36/EGFR co-localization studies

  3. Viral infection models:

     • Study viral hijacking of ESCRT machinery using VPS36 antibodies

     • Track changes in ESCRT-II complex formation during viral replication

     • The coronavirus RTC-proximal proteome has identified ESCRT components, suggesting viral interaction with this pathway

  4. Methodological considerations for disease models:

     • Use biotin-conjugated VPS36 antibody at 1:50-1:200 for immunofluorescence

     • For tissue sections, include antigen retrieval with TE buffer pH 9.0

     • In multiplex studies, combine with disease-specific markers

     • For quantification, employ high-content imaging with automated analysis

  5. Validation in human samples:

     • Human pancreatic cancer and intrahepatic cholangiocarcinoma tissues have been validated for VPS36 immunohistochemistry

     • Western blot applications have been validated across multiple human cell lines

  These applications connect fundamental ESCRT pathway mechanisms to clinically relevant disease processes, offering insights into potential therapeutic targets within the ESCRT machinery.