VPS4B Antibody

What is VPS4B and why are antibodies against it important in research?

VPS4B (also known as MIG1, SKD1, SKD1B, or VPS4-2) is a member of the AAA ATPase protein family involved in critical cellular membrane remodeling processes. This 444-amino acid protein (49.3 kDa) functions in late steps of the endosomal multivesicular bodies (MVB) pathway .

VPS4B antibodies are essential research tools because they enable:

• Tracking of endogenous VPS4B in various cellular compartments

• Investigation of ESCRT machinery assembly/disassembly cycles

• Examination of multivesicular body formation mechanisms

• Study of viral budding processes that hijack ESCRT machinery

• Exploration of VPS4B involvement in cancer progression

When selecting VPS4B antibodies, researchers should consider the specific applications needed (WB, IHC, ICC/IF), species reactivity requirements, and whether monoclonal specificity or polyclonal broad epitope recognition would better serve their research goals .

What are the critical differences between VPS4A and VPS4B that researchers should know?

Despite their functional similarity, VPS4A and VPS4B exhibit important distinctions that affect experimental design:

Importantly, depleting one paralog does not affect the expression level of the other, suggesting independent regulation mechanisms . This characteristic makes them particularly interesting for synthetic lethality studies, where targeting VPS4A in VPS4B-deficient cancer cells shows therapeutic potential .

Methods to distinguish between these paralogs include:

• Using highly specific antibodies validated against knockout controls

• Employing paralog-specific siRNAs/shRNAs with validated specificity

• Designing primers for RT-qPCR that target non-homologous regions

What applications are VPS4B antibodies most commonly used for, and what are the optimal conditions?

VPS4B antibodies are versatile tools applicable across multiple techniques, with specific optimization parameters:

Each application requires specific optimization:

• For WB: Use 5% BSA for blocking to minimize background

• For IP: Pre-clear lysates thoroughly to reduce non-specific binding

• For IF: Counter-stain with endosomal markers (EEA1, CD63) to confirm localization

• For IHC: Validate staining patterns using known positive (appendix) and negative (muscle) tissues

How should researchers troubleshoot inconsistent VPS4B antibody staining patterns?

When facing variable VPS4B staining results, systematic troubleshooting is essential:

• Antibody validation concerns:

  • Confirm antibody specificity using VPS4B-knockout controls

  • Test multiple antibodies targeting different epitopes

  • Verify lot-to-lot consistency with standard positive controls

• Sample preparation issues:

  • For IHC: Standardize fixation protocols (duration, fixative concentration)

  • For WB: Ensure consistent lysis conditions that preserve protein integrity

  • For IF: Optimize fixation and permeabilization for endosomal proteins

• Technical considerations:

  • Standardize incubation times and temperatures

  • Test a range of antibody concentrations (titration series)

  • For IHC: Perform systematic antigen retrieval optimization

• Biological variables:

  • Cell cycle fluctuations can affect VPS4B expression/localization

  • Stress conditions alter MVB pathway activity

  • Endosomal dynamics change with cellular state

When optimizing, create a systematic testing matrix documenting all variables. The Human Protein Atlas project has validated select VPS4B antibodies for tissue staining, providing useful reference staining patterns .

What experimental design considerations are important when studying VPS4B's ATPase activity?

VPS4B's ATPase activity is central to its function in ESCRT complex disassembly. Proper experimental design requires:

• Assay selection:

  • ADP² assay reliably measures ADP produced by VPS4B enzymatic activity

  • Compatible with 96, 384, and 1536-well formats for higher throughput

• Reaction optimization:

  • Buffer composition significantly impacts activity: 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 50 mM KCl, 5 mM DTT, and 0.01% Triton

  • Temperature control (37°C) critical for reproducible results

  • Reaction time (typically two hours) affects sensitivity

• Data analysis considerations:

  • Convert raw data to ADP using standard curves

  • Evaluate linearity under initial velocity conditions

  • Calculate Z' factors to assess assay robustness (Z' > 0.5 indicates suitability for HTS)

• Controls:

  • Include ATP-only and enzyme-only controls

  • Use known ATPase inhibitors as positive controls

  • Include ADP standard curves in each experiment

The BellBrook Labs VPS4B assay demonstrates excellent performance with Z' values of 0.81, indicating high reliability for inhibitor screening applications .

How can researchers design experiments to investigate synthetic lethality between VPS4A and VPS4B?

The synthetic lethality between VPS4A and VPS4B offers promising therapeutic opportunities for VPS4B-deficient cancers. Rigorous experimental design should include:

• Cell model selection:

  • Use isogenic cell line pairs (e.g., HCT116 VPS4B⁻/⁻ and parental lines)

  • Screen cancer cell lines for natural VPS4B deficiency

  • Create doxycycline-inducible shRNA systems for controlled depletion

• Knockdown/knockout strategies:

  • Validate siRNA/shRNA specificity for each paralog

  • Confirm knockdown at both mRNA and protein levels

  • Use multiple independent siRNA sequences to rule out off-target effects

• Viability and phenotypic assessments:

  • Compare single vs. dual knockdown effects on cell viability

  • Evaluate morphological changes in endosomal compartments

  • Assess cellular transcriptome alterations

• In vivo validation:

  • Establish xenograft models with inducible systems

  • Monitor tumor growth upon conditional VPS4A depletion in VPS4B-deficient tumors

  • Analyze immune response markers in the tumor microenvironment

• Mechanistic investigations:

  • Analyze the release of immunomodulatory molecules

  • Evaluate indicators of immunogenic cell death

  • Study ESCRT dysfunction consequences

The Mami et al. (2020) study demonstrated that VPS4A depletion in VPS4B-deficient colorectal cancer cells induced profound transcriptome alterations leading to immunogenic cell death, providing a model experimental approach .

What are the best practices for validating VPS4B antibodies for experimental use?

Thorough validation is essential before employing VPS4B antibodies in critical experiments:

• Specificity testing:

  • Test on VPS4B knockout/knockdown samples

  • Verify absence of cross-reactivity with VPS4A (despite ~80% homology)

  • Use peptide competition assays to confirm epitope specificity

• Multi-technique validation:

  • Confirm consistent results across different applications (WB, IF, IHC)

  • Compare staining patterns with published subcellular localization data

  • Validate in multiple cell types with known VPS4B expression levels

• Reference tissue validation:

  • Test on tissues with known high expression (e.g., appendix) and low expression (e.g., muscle)

  • Compare with staining patterns in Human Protein Atlas

  • Evaluate batch-to-batch consistency using standard tissue samples

• Functional validation:

  • Confirm ability to detect VPS4B in immunoprecipitation of ESCRT complexes

  • Verify expected molecular weight (49-50 kDa) in Western blots

  • Test ability to detect endosomal localization in immunofluorescence

For IHC applications, semi-quantitative scoring based on staining intensity provides reliable assessment of VPS4B abundance across tissue samples .

What experimental approaches can effectively study interactions between VPS4B and other ESCRT components?

Investigating VPS4B-ESCRT interactions requires sophisticated methodological approaches:

• Co-immunoprecipitation strategies:

  • Use crosslinking to capture transient interactions

  • Employ sequential IPs to isolate specific subcomplexes

  • Optimize lysis conditions to preserve membrane-associated complexes

• Proximity labeling techniques:

  • BioID or APEX2 fusions to VPS4B capture interaction partners

  • TurboID provides faster labeling for dynamic ESCRT assemblies

  • Analyze labeled proteins by mass spectrometry

• Live-cell imaging approaches:

  • Fluorescently tagged VPS4B (maintaining ATPase activity)

  • FRET-based sensors to detect conformational changes

  • Dual-color imaging to track VPS4B and ESCRT-III recruitment dynamics

• In vitro reconstitution:

  • Purified components on model membranes

  • GUV-based assays to visualize membrane deformation

  • ATP-dependent assembly/disassembly cycles

• Functional mutant analysis:

  • ATP-binding mutants (K180A) for dominant-negative effects

  • MIT domain mutants to disrupt ESCRT-III binding

  • Compare with VPS4A equivalent mutations

These approaches have revealed that VPS4B recognizes membrane-associated ESCRT-III assemblies and catalyzes their ATP-dependent disassembly, potentially in combination with membrane fission .

How should researchers interpret VPS4B expression data in cancer samples?

VPS4B expression analysis in cancer requires careful consideration of multiple factors:

• Methodological considerations:

  • Compare protein (IHC/WB) and mRNA expression levels

  • Use semi-quantitative scoring methods based on staining intensity

  • Consider tissue microarrays for high-throughput analysis

• Data interpretation framework:

  • Analyze matched normal-tumor pairs from the same patient

  • Stratify by cancer subtype and stage

  • Correlate with clinical outcomes data

• Technical validation:

  • Validate antibody specificity in cancer tissues

  • Use multiple antibodies targeting different epitopes

  • Confirm with orthogonal methods (RT-qPCR, proteomics)

• Biological context:

  • Evaluate VPS4A/VPS4B ratio rather than absolute levels

  • Consider compensatory mechanisms

  • Analyze entire ESCRT pathway component expression

The study by Mami et al. demonstrated that VPS4B downregulation in colorectal cancer corresponded to decreased VPS4B protein abundance using tissue microarrays of 100 pairs of matched normal colon and treatment-naïve primary CRC samples . This systematic approach represents best practices for cancer expression studies.

What strategies help researchers evaluate VPS4B's role in viral budding mechanisms?

VPS4B's involvement in viral budding requires specialized experimental approaches:

• Cellular models:

  • Inducible VPS4B dominant-negative systems

  • VPS4B knockout/knockdown in permissive cell lines

  • Complementation with mutant VPS4B variants

• Viral budding assays:

  • Quantification of extracellular vs. cell-associated virus

  • Single-particle tracking of viral egress

  • Electron microscopy to visualize budding arrest phenotypes

• ESCRT recruitment analysis:

  • Immunofluorescence co-localization with viral assembly sites

  • Live-cell imaging of fluorescently tagged VPS4B during infection

  • Biochemical fractionation of membrane-associated complexes

• Structure-function studies:

  • ATPase mutants to dissect energy-dependent steps

  • MIT domain mutants to disrupt ESCRT-III interactions

  • Chimeric VPS4A/B proteins to identify paralog-specific functions

VPS4B works with the ESCRT machinery in topologically equivalent membrane fission events during viral budding (particularly HIV-1 and other lentiviruses) , making it an important target for antiviral research.

What are the most reliable methods for quantifying VPS4B protein levels across different sample types?

Accurate VPS4B quantification requires technique-specific optimizations:

• Western blot quantification:

  • Use validated antibodies with demonstrated specificity

  • Include recombinant VPS4B standard curves

  • Normalize to multiple housekeeping proteins (GAPDH, β-actin, tubulin)

  • Apply densitometric analysis with linear dynamic range validation

• Mass spectrometry approaches:

  • Selected reaction monitoring (SRM) for targeted quantification

  • Heavy-labeled peptide standards for absolute quantification

  • Parallel reaction monitoring for increased specificity

  • Focus on unique peptides not shared with VPS4A

• Flow cytometry:

  • Intracellular staining protocols optimized for permeabilization

  • Validation with positive and negative controls

  • Multi-parameter analysis with endosomal markers

• ELISA/immunoassays:

  • Sandwich assays with non-competing antibody pairs

  • Recombinant protein standard curves

  • Spike-in recovery tests for matrix effects

• Immunohistochemistry quantification:

  • Digital pathology platforms for standardized scoring

  • Semi-quantitative intensity scoring (0-3 scale)

  • Automated image analysis with region-specific quantification

For cell line work, Western blotting using antibodies like Proteintech's 68443-1-Ig has demonstrated consistent detection of the ~50 kDa VPS4B band across multiple cell types including LNCaP, NIH/3T3, HeLa, HEK-293, Jurkat, K-562, and HSC-T6 cells .

How can researchers optimize experimental design to study VPS4B's role in multivesicular body formation?

Investigating VPS4B's function in MVB biogenesis requires specialized approaches:

• Cellular systems:

  • Inducible VPS4B depletion/expression

  • ATP-locked VPS4B mutants (E235Q)

  • Cell types with prominent MVB machinery (e.g., macrophages, B cells)

• Ultrastructural analysis:

  • Electron microscopy to visualize MVB morphology

  • Correlative light-electron microscopy linking fluorescent markers to ultrastructure

  • Immunogold labeling for VPS4B localization

• Cargo trafficking assays:

  • EGFR degradation kinetics following EGF stimulation

  • Lysosomal hydrolase sorting pathways

  • Exosomal marker (CD63, SDCBP) release quantification

• Live-cell imaging approaches:

  • Super-resolution microscopy of endosomal dynamics

  • Fluorescent pH sensors to monitor endosomal maturation

  • Dual-color imaging of VPS4B and cargo proteins

• Biochemical fractionation:

  • Isolation of MVBs using density gradients

  • Immuno-isolation of specific endosomal populations

  • Proteomic analysis of MVB-enriched fractions

VPS4B is directly involved in the redistribution of ESCRT-III components to the cytoplasm for further rounds of MVB sorting, making its activity central to the process of intraluminal vesicle formation .