VPS4B Antibody, Biotin conjugated

What is VPS4B and why is it important in cellular research?

VPS4B (Vacuolar Protein Sorting 4 Homolog B) is a critical component in the endosomal multivesicular bodies (MVB) pathway. In humans, it's a protein with 444 amino acid residues and a molecular mass of 49.3 kDa, belonging to the AAA ATPase protein family . It recognizes membrane-associated ESCRT-III assemblies and catalyzes their disassembly, possibly in combination with membrane fission. VPS4B redistributes the ESCRT-III components to the cytoplasm for further rounds of MVB sorting . Its importance in research stems from its role in fundamental cellular processes including receptor degradation, membrane protein trafficking, cytokinesis, and viral budding, making it a key target for studies in cellular biology, virology, and disease mechanisms.

How does biotin conjugation affect VPS4B antibody applications?

Biotin conjugation significantly enhances the versatility of VPS4B antibodies by enabling signal amplification through the strong biotin-avidin/streptavidin interaction. Unlike unconjugated antibodies, biotin-conjugated VPS4B antibodies allow for enhanced sensitivity in detection methods, particularly in ELISA applications . The biotin tag enables multiple detection options, including colorimetric, fluorescent, and chemiluminescent readouts when combined with appropriate avidin-conjugated reporter molecules. This conjugation preserves antibody specificity while providing greater flexibility in experimental design and detection strategies. Researchers should note that while biotin conjugation increases detection sensitivity, it may occasionally affect epitope recognition or introduce steric hindrance in certain applications, necessitating validation for specific experimental contexts.

What are the optimal storage conditions for VPS4B antibody, biotin conjugated?

For maximum stability and activity retention, VPS4B antibody, biotin conjugated should be stored in 50% glycerol with 0.01M PBS at pH 7.4, containing 0.03% Proclin 300 as a preservative . Storage temperature should be maintained at -20°C for long-term preservation, avoiding repeated freeze-thaw cycles which can degrade both antibody activity and the biotin conjugation. Aliquoting the antibody upon receipt is strongly recommended to minimize freeze-thaw cycles. Short-term storage (1-2 weeks) at 4°C is acceptable, but extended storage at this temperature may result in decreased activity and potential microbial contamination. Exposure to direct light should be minimized, particularly for working solutions, as photobleaching can affect biotin-conjugated detection systems.

How can VPS4B antibody be used to study ESCRT-III interactions?

VPS4B antibody can be leveraged to investigate ESCRT-III (Endosomal Sorting Complex Required for Transport-III) interactions through several advanced approaches. Immunoprecipitation followed by mass spectrometry can identify novel VPS4B binding partners within the ESCRT machinery . Researchers can employ proximity ligation assays (PLA) using biotin-conjugated VPS4B antibody alongside antibodies against suspected interaction partners to visualize protein-protein interactions in situ. The biotin conjugation provides an amplification advantage through streptavidin-based detection systems. Co-immunofluorescence studies can reveal spatial relationships between VPS4B and ESCRT-III components during membrane scission events. For dynamic studies, VPS4B antibody can be used to track the temporal recruitment of ESCRT machinery during processes like viral budding or cytokinesis. When designing these experiments, researchers should include appropriate controls to confirm specificity, such as VPS4B knockdown samples and isotype controls.

What are the comparative detection limits between different VPS4B antibody formats?

The detection sensitivity of VPS4B antibodies varies considerably across different formats and applications. Based on research data, biotin-conjugated VPS4B antibodies demonstrate approximately 2-5 fold higher sensitivity in ELISA applications compared to unconjugated formats . The table below summarizes comparative detection limits:

Researchers should note that biotin-conjugated formats excel in ELISA and specialized applications requiring signal amplification, while direct conjugates (HRP, fluorophores) may be preferred for more straightforward detection methods. Detection limits can vary based on experimental conditions, sample preparation, and antibody lot-to-lot variations, necessitating standardization for quantitative comparisons.

How can VPS4B antibody, biotin conjugated be used to study viral budding mechanisms?

VPS4B antibody, biotin conjugated, serves as a powerful tool for investigating viral budding mechanisms, particularly for enveloped viruses like HIV-1 and other lentiviruses that hijack the ESCRT machinery. Researchers can employ this antibody in immunofluorescence studies to visualize VPS4B recruitment to viral assembly sites during the late stages of infection . The biotin conjugation enables signal amplification through streptavidin-based detection systems, enhancing sensitivity for detecting potentially low-abundance VPS4B at budding sites. Co-immunoprecipitation experiments can identify viral proteins that interact with VPS4B during budding. For quantitative analysis, researchers can design time-course experiments using the antibody to track VPS4B localization throughout the viral life cycle, correlating its recruitment with specific stages of virion assembly and release. When designing these studies, it's crucial to include appropriate controls, such as VPS4B-depleted cells or cells expressing dominant-negative VPS4B mutants, to confirm the specificity of the observed interactions and their functional relevance to viral budding.

What protocol modifications are recommended for using VPS4B antibody in different applications?

Optimizing VPS4B antibody protocols requires specific modifications based on the intended application. For Western blotting applications, researchers should use a 1:500-1:1000 dilution of the antibody, with overnight incubation at 4°C to maximize specific binding . Including 5% BSA rather than milk-based blocking buffers can reduce background when detecting phosphorylated forms of VPS4B. For immunofluorescence, a higher dilution (1:100-1:200) is typically recommended, with extended primary antibody incubation (overnight at 4°C) and thorough washing steps to minimize background. When using biotin-conjugated VPS4B antibody in ELISA applications, optimization of both antibody concentration (typically 1:100-1:500) and streptavidin-HRP dilution is critical for achieving maximum signal-to-noise ratio . For immunoprecipitation studies, pre-clearing lysates with protein A/G beads before adding the VPS4B antibody significantly reduces non-specific binding. In multi-parameter flow cytometry, careful titration of the antibody is essential to determine optimal staining concentration while avoiding fluorescence spillover into other channels.

What are the key troubleshooting approaches for inconsistent VPS4B antibody staining?

When encountering inconsistent VPS4B antibody staining, researchers should systematically address several potential issues. First, verify antibody integrity by checking storage conditions and expiration dates, as improper handling can lead to degradation of both the antibody and biotin conjugation . For weak signals, consider implementing signal amplification strategies such as tyramide signal amplification (TSA) or polymer-based detection systems compatible with biotin-conjugated antibodies. High background issues can often be resolved by optimizing blocking conditions (extending blocking time or using different blocking agents) and increasing wash duration and frequency. For inconsistent cell-to-cell staining in immunofluorescence, evaluate fixation methods, as overfixation can mask epitopes while underfixation may cause antigen leakage. Cross-reactivity problems can be addressed by performing pre-absorption with the immunizing peptide or using more stringent washing conditions. When troubleshooting biotin-conjugated VPS4B antibody specifically, consider that endogenous biotin in certain tissues may contribute to background; this can be mitigated using commercial biotin-blocking kits prior to antibody application.

How should researchers quantify VPS4B expression levels across different experimental conditions?

Accurate quantification of VPS4B expression requires standardized approaches adapted to specific experimental contexts. For Western blot analysis, normalization to housekeeping proteins (such as GAPDH, β-actin, or α-tubulin) is essential for reliable comparisons across conditions . Densitometric analysis should include multiple biological replicates (minimum n=3) and technical replicates to ensure statistical validity. For immunofluorescence quantification, researchers should employ automated image analysis platforms with consistent threshold settings across all experimental conditions. Cell-by-cell quantification using single-cell analysis approaches provides more granular data than whole-field measurements, particularly in heterogeneous populations. When using flow cytometry for VPS4B quantification, calibration beads should be included to convert fluorescence intensity into standardized units (molecules of equivalent soluble fluorochrome, MESF), enabling cross-experimental comparisons. For ELISA-based quantification with biotin-conjugated VPS4B antibody, a standard curve using recombinant VPS4B protein at known concentrations is essential for accurate determination of absolute protein levels . Regardless of the method chosen, researchers should include appropriate positive and negative controls, including VPS4B knockdown or knockout samples when available.

How does VPS4B dysfunction impact cellular homeostasis and disease mechanisms?

VPS4B dysfunction significantly disrupts cellular homeostasis through multiple interconnected pathways. As an essential component of the ESCRT machinery, VPS4B impairment leads to defective multivesicular body formation, resulting in abnormal receptor recycling and degradation . This can cause sustained receptor signaling, particularly for growth factor receptors, potentially contributing to carcinogenesis. Research data indicates that VPS4B dysfunction disturbs autophagic flux, as MVBs are critical intermediates in autophagosome-lysosome fusion; this has implications for neurodegenerative disorders where protein aggregation is a hallmark feature. In the context of viral infections, aberrant VPS4B function can either enhance or inhibit viral budding depending on the specific virus, as many enveloped viruses hijack the ESCRT machinery for efficient release . VPS4B dysregulation has also been implicated in cytokinesis defects, leading to genomic instability and aneuploidy. Recent studies suggest connections between VPS4B abnormalities and exosome composition alterations, potentially affecting intercellular communication pathways relevant to immune responses and cancer progression. When interpreting disease-related VPS4B data, researchers should consider its context-dependent functions and interaction with various ESCRT-III components.

What are the reported expression patterns of VPS4B across different cell types and tissues?

VPS4B demonstrates complex expression patterns that vary across tissues and developmental stages. According to comprehensive immunohistochemical studies, VPS4B is ubiquitously expressed across many tissue types, with particularly high expression in metabolically active tissues . The table below summarizes VPS4B expression patterns based on published research:

Developmentally, VPS4B expression is dynamically regulated, with significant upregulation during embryonic development stages involving extensive membrane remodeling. In pathological contexts, altered VPS4B expression has been documented in multiple cancer types, neurodegenerative conditions, and during viral infections. These expression patterns highlight the context-specific roles of VPS4B and provide important baseline information for researchers investigating its function in specific tissues or disease states.

What emerging technologies are advancing VPS4B research beyond traditional antibody applications?

Cutting-edge technologies are revolutionizing VPS4B research beyond conventional antibody-based approaches. CRISPR-Cas9 gene editing now enables precise manipulation of VPS4B through knockout, knockin, and point mutations to study function in endogenous contexts . Proximity labeling techniques like BioID and APEX2 are being applied to VPS4B to map its protein interaction network with temporal and spatial resolution. Super-resolution microscopy approaches (STORM, PALM, STED) overcome the diffraction limit to visualize VPS4B dynamics at nanoscale resolution, revealing previously undetectable structural arrangements during ESCRT-mediated membrane remodeling. Researchers are developing split-fluorescent protein systems with VPS4B to monitor protein-protein interactions in real-time. Cryo-electron microscopy and tomography are providing structural insights into VPS4B-ESCRT-III complexes during membrane scission events. Single-molecule tracking approaches are being applied to follow individual VPS4B molecules during endosomal sorting and viral budding. Mass spectrometry-based proteomics, particularly combined with proximity labeling, is enabling comprehensive characterization of VPS4B interactomes under various physiological and pathological conditions . These emerging technologies complement traditional antibody-based approaches and are advancing our understanding of VPS4B's multifaceted roles in cellular homeostasis.

How does VPS4B research contribute to therapeutic development for diseases involving membrane trafficking defects?

VPS4B research offers significant potential for therapeutic interventions targeting diseases with membrane trafficking abnormalities. By understanding VPS4B's role in receptor degradation pathways, researchers are developing strategies to modulate growth factor receptor signaling in cancers where these pathways are dysregulated . VPS4B's critical function in viral budding makes it an attractive target for broad-spectrum antiviral therapeutics, with small molecule inhibitors of VPS4B ATPase activity showing promise in preliminary studies against enveloped viruses including HIV and SARS-CoV-2. In neurodegenerative diseases characterized by protein aggregation, strategies to enhance VPS4B function may promote clearance of toxic protein species through improved endolysosomal degradation. The role of VPS4B in exosome biogenesis has implications for immunotherapy approaches, as manipulating VPS4B activity could potentially alter exosome cargo and immunomodulatory properties. High-throughput screening approaches using VPS4B antibodies are identifying compounds that specifically modulate its interactions with ESCRT-III components, opening avenues for targeted therapeutic development. As research progresses, understanding the tissue-specific functions and regulatory mechanisms of VPS4B will be crucial for developing therapies with acceptable safety profiles, as complete inhibition may have deleterious effects on fundamental cellular processes.