vps26bl Antibody

What is VPS26B and what cellular functions does it perform?

VPS26B (Vacuolar protein sorting-associated protein 26B) acts as a core component of the retromer cargo-selective complex (CSC). This complex prevents the missorting of selected transmembrane cargo proteins into lysosomal degradation pathways . The CSC, including VPS26B, is recruited to endosomal membranes through interactions with RAB7A and SNX3 . VPS26B participates in various trafficking pathways: the SNX-BAR retromer mediates retrograde transport from endosomes to the trans-Golgi network (TGN), the SNX3-retromer mediates retrograde transport of WLS, and the SNX27-retromer is involved in endosome-to-plasma membrane trafficking . Functionally, VPS26B acts redundantly with VPS26A in some contexts, particularly in SNX-27 mediated endocytic recycling of proteins like SLC2A1/GLUT1 . Understanding these functions provides the foundation for designing experiments that investigate intracellular trafficking mechanisms.

What are the common applications for VPS26B antibodies in research?

VPS26B antibodies serve multiple research applications with varying methodological considerations. Western blotting is a primary application, with recommended dilutions of 1:500-1:1000 for optimal detection of the approximately 39kDa VPS26B protein . Immunohistochemistry on paraffin-embedded tissues (IHC-P) enables visualization of VPS26B in tissue contexts, while immunocytochemistry and immunofluorescence (ICC/IF) allow for subcellular localization studies . Immunoprecipitation (IP) can be employed to isolate VPS26B and its interacting partners, offering insights into protein complexes . ELISA applications are also supported, typically at higher dilutions (1:10000) . Researchers should optimize conditions for each application, as antibody performance may vary by experimental context and sample type. Validation using appropriate controls is essential for generating reliable scientific data.

How does VPS26B relate to VPS26A, and can antibodies cross-react?

VPS26A and VPS26B are paralogous proteins that share functional similarities as components of the retromer complex. Despite their shared roles, they exhibit distinct expression patterns and potentially specialized functions in different cellular contexts . VPS26A has been implicated in pancreatic adenocarcinoma (PAAD), where its elevated expression correlates with histological type, tumor stage, and poor prognosis . Unlike VPS26B, VPS26A expression significantly correlates with immune infiltration in PAAD, particularly with B cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells .

What experimental controls are critical when validating VPS26B antibody specificity?

Rigorous validation of VPS26B antibody specificity requires multiple complementary approaches. The primary control is the peptide competition assay, where parallel samples are probed with antibody either alone or pre-incubated with the immunizing peptide . Disappearance of bands in the competitive condition confirms specificity, as demonstrated in western blot validation data from commercial antibodies .

For genetic validation, CRISPR/Cas9 knockout or siRNA knockdown of VPS26B provides the gold standard control. In these experiments, antibody reactivity should be absent or significantly reduced in knockout/knockdown samples compared to wild-type controls. When using multiple antibodies targeting different epitopes of VPS26B, concordant results strengthen validity.

How can researchers distinguish between retromer subcomplexes that contain VPS26B versus VPS26A?

Distinguishing between retromer subcomplexes containing either VPS26B or VPS26A requires sophisticated biochemical and imaging approaches. Immunoprecipitation with antibodies specific to VPS26B (using validated antibodies like A12325-2 or ab236966) followed by mass spectrometry can identify differential interactors specific to VPS26B-containing complexes. Sequential immunoprecipitation, where samples are first depleted of VPS26A-containing complexes before isolating VPS26B complexes, can provide cleaner separation.

For imaging-based approaches, dual-color immunofluorescence with specific antibodies against VPS26A and VPS26B, combined with super-resolution microscopy techniques like STORM or STED, can resolve distinct subpopulations. Co-localization analysis with subcomplex-specific markers (e.g., SNX27 for SNX27-retromer) can further define functional differences.

Functionally, cargo-specific trafficking assays using fluorescently tagged cargo proteins known to preferentially interact with either VPS26A or VPS26B can reveal distinct roles. Comparative quantification following selective depletion of either paralogue through siRNA or CRISPR approaches provides additional discrimination. For retromer assembly studies, in vitro reconstitution experiments with purified components can establish whether VPS26B forms complexes with different affinities or conformational properties compared to VPS26A-containing complexes.

What methodological approaches can identify novel VPS26B interacting partners?

Identifying novel VPS26B interacting partners requires a multi-tiered approach combining complementary techniques. Proximity-dependent biotin labeling (BioID or TurboID) with VPS26B fusion proteins represents a powerful approach for capturing transient or weak interactions in living cells. This technique involves expressing VPS26B fused to a biotin ligase, allowing biotinylation of proteins in close proximity, followed by streptavidin pulldown and mass spectrometry identification.

Conventional co-immunoprecipitation using validated VPS26B antibodies under varying detergent and salt conditions can capture stable interactions. Crosslinking mass spectrometry (XL-MS) provides additional structural information by covalently linking interacting proteins prior to digestion and analysis, revealing spatial relationships within complexes.

For dynamic interactions, fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) with VPS26B fusion constructs allow real-time visualization of protein interactions in living cells. Split-luciferase complementation assays provide quantitative measurement of interaction strengths.

Yeast two-hybrid screening with VPS26B as bait against tissue-specific libraries can identify direct binding partners. Validation of identified interactions should include reverse co-immunoprecipitation, domain mapping through truncation mutants, and functional studies using siRNA knockdown of putative partners. Correlation analysis of expression patterns across tissues and disease states can provide contextual relevance for newly identified interactions.

How does VPS26B expression correlate with immune infiltration in cancer contexts?

Unlike VPS26A, which shows significant correlation with immune infiltration in pancreatic adenocarcinoma (PAAD), the specific relationship between VPS26B and immune infiltration in cancer has not been comprehensively characterized in the provided search results . For comparison, VPS26A expression positively correlates with infiltration of B cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells in PAAD according to TIMER database analysis .

To investigate potential VPS26B-immune correlations, researchers should employ similar methodological approaches: analysis of TCGA datasets using computational tools like TIMER, CIBERSORT, or xCell to quantify immune cell infiltration in relation to VPS26B expression across cancer types. Single-cell RNA sequencing of tumor microenvironments would provide higher resolution of immune cell subtypes correlated with VPS26B expression.

Experimental validation would require tissue microarray immunostaining for VPS26B and immune cell markers in cancer cohorts, with multiplex immunofluorescence enabling spatial relationship analysis. Functionally, co-culture experiments with VPS26B-modulated cancer cells and immune cells (T cells, macrophages) could reveal direct effects on immune function. Mechanistic studies might investigate whether VPS26B affects trafficking of immune modulatory receptors or secretion of chemokines that influence immune recruitment. This systematic approach would establish whether VPS26B, like VPS26A, plays a role in cancer immunobiology.

What are the optimal conditions for using VPS26B antibodies in western blotting applications?

Optimal western blotting with VPS26B antibodies requires careful consideration of several technical parameters. Sample preparation should include complete lysis using RIPA or NP-40 buffer supplemented with protease inhibitors, with 20-40μg of total protein loaded per lane. For VPS26B detection, 10-12% polyacrylamide gels provide optimal resolution around the 39kDa range where VPS26B migrates .

After transfer to PVDF or nitrocellulose membranes, blocking should be performed with 5% non-fat milk or BSA in TBS-T for 1 hour at room temperature. VPS26B antibodies should be diluted in blocking buffer at 1:500-1:1000 for primary antibody incubation , with overnight incubation at 4°C providing optimal signal-to-noise ratio. For secondary detection, anti-rabbit HRP-conjugated antibodies at 1:5000-1:10000 perform well with standard ECL detection systems.

Critical controls include peptide competition assays, where parallel membranes are probed with antibody pre-incubated with immunizing peptide . Positive controls should include cell lines known to express VPS26B (LOVO cells have been validated in published data) . Stripping and reprobing for housekeeping proteins like β-actin or GAPDH ensures equal loading. When detecting both VPS26A and VPS26B in the same experiment, careful antibody selection and validation is essential to avoid cross-reactivity between these paralogues.

What are the key considerations for immunohistochemistry applications with VPS26B antibodies?

Successful immunohistochemistry with VPS26B antibodies requires optimization of several critical parameters. Tissue fixation should be standardized, typically using 10% neutral buffered formalin for 24-48 hours. Antigen retrieval is essential due to formalin-induced crosslinking, with heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) recommended for VPS26B detection.

Blocking endogenous peroxidases (3% H₂O₂) and non-specific binding (5-10% normal goat serum) minimizes background. VPS26B antibody concentration requires titration for each application, starting with manufacturer recommendations and adjusting as needed . Overnight incubation at 4°C generally provides optimal sensitivity and specificity. Detection systems should match the primary antibody host; for rabbit polyclonal VPS26B antibodies, polymer-based detection systems offer high sensitivity with low background.

Essential controls include negative controls (primary antibody omission and isotype controls) , positive controls (tissues with known VPS26B expression), and peptide competition controls. For multiplex staining with other retromer components, sequential staining protocols with appropriate antibody stripping between rounds minimizes cross-reactivity.

For quantification, digital image analysis using standardized thresholds helps ensure reproducibility. Subcellular localization assessment should consider VPS26B's expected punctate cytoplasmic distribution consistent with endosomal localization. When comparing normal versus pathological tissues, standardization of all staining conditions and processing times is critical for valid comparisons.

How can researchers troubleshoot poor signal or high background when using VPS26B antibodies?

Troubleshooting poor signal or high background with VPS26B antibodies requires systematic assessment of multiple technical factors. For weak signals in western blotting, researchers should first verify protein loading (30-50μg typically provides detectable VPS26B signal) and transfer efficiency using reversible stains like Ponceau S. Fresh antibody preparations and extended exposure times may enhance detection. Primary antibody concentration can be increased (up to 1:250) , with overnight incubation at 4°C. Enhanced chemiluminescence (ECL) substrates with higher sensitivity or signal amplification systems may be required for low-abundance samples.

For high background, optimizing blocking conditions is critical. Extended blocking (2 hours at room temperature) with 5% BSA or 5% non-fat milk, and including 0.05-0.1% Tween-20 in wash buffers reduces non-specific binding. Reducing secondary antibody concentration and adding 0.1-0.3M NaCl to antibody dilution buffers can minimize ionic interactions contributing to background.

In immunohistochemistry or immunofluorescence applications, background issues may result from inadequate blocking of endogenous peroxidases, biotin, or antibody-binding proteins. Pre-absorption of antibodies with tissue lysates from species matching the experimental samples can reduce non-specific binding. For formalin-fixed tissues, optimizing antigen retrieval conditions through time and pH titration is often critical for specific signal enhancement.

Cross-reactivity with VPS26A can be assessed through peptide competition assays with both VPS26A and VPS26B peptides separately. If signals persist despite optimization, alternative VPS26B antibodies targeting different epitopes should be evaluated to confirm findings.

What is the role of VPS26B in neurodegenerative disorders?

While the specific role of VPS26B in neurodegenerative disorders is not directly addressed in the provided search results, methodological approaches to investigate this question can be derived from retromer biology knowledge. As a component of the retromer cargo-selective complex, VPS26B likely contributes to protein trafficking pathways essential for neuronal health . Researchers should examine VPS26B expression and localization in relevant neuronal populations using immunohistochemistry with validated VPS26B antibodies on post-mortem brain tissue from neurodegenerative disease patients versus controls .

Functional studies could involve manipulating VPS26B levels in neuronal cell models through CRISPR/Cas9 or shRNA approaches, followed by assessment of effects on trafficking of neurodegeneration-associated cargoes like APP, SORL1, or tau. Proximity labeling techniques with VPS26B as bait could identify neuron-specific interaction partners that might be compromised in disease states.

Mouse models with conditional VPS26B knockout in specific neuronal populations would allow evaluation of age-dependent neurodegeneration phenotypes. Single-cell transcriptomics comparing VPS26B expression across brain cell types in health and disease could identify vulnerable populations. Proteomics of VPS26B-containing complexes isolated from control versus diseased brain tissue might reveal altered interactions or post-translational modifications affecting function.

For direct disease relevance, genetic association studies should examine whether VPS26B variants correlate with neurodegenerative disease risk or progression. The methodological integration of these approaches would establish whether VPS26B, like other retromer components, plays a causal role in neurodegeneration or represents a potential therapeutic target.

How can researchers measure VPS26B-mediated trafficking in live cells?

Measuring VPS26B-mediated trafficking in live cells requires sophisticated imaging techniques combined with cargo-specific assays. Researchers should begin by generating fluorescently tagged VPS26B constructs (e.g., VPS26B-GFP) expressed at near-endogenous levels, ideally using CRISPR knock-in approaches to maintain physiological regulation. Time-lapse confocal microscopy with these constructs enables tracking of VPS26B-positive endosomal structures in real-time.

For cargo-specific trafficking assays, fluorescently tagged reporters known to utilize retromer for recycling (e.g., CI-MPR, sortilin, SorLA) can be co-expressed and their co-localization with VPS26B monitored over time. Pulse-chase protocols with photoactivatable or photoconvertible cargo proteins allow precise temporal control over trafficking cohorts. Surface biotinylation assays combined with immunoprecipitation using VPS26B antibodies can biochemically quantify cargo recycling rates.

RUSH (Retention Using Selective Hooks) systems provide temporal control over cargo release from the ER, allowing synchronized trafficking through the secretory and endocytic pathways. pH-sensitive fluorescent cargo constructs can distinguish between endosomal compartments based on luminal acidity. For higher resolution, lattice light-sheet microscopy or TIRF microscopy enables visualization of vesicle formation and scission events at the plasma membrane or endosomal surfaces.

Functional disruption through VPS26B siRNA knockdown or expression of dominant-negative mutants allows assessment of VPS26B's specific contribution to trafficking pathways. Quantification should include parameters such as vesicle velocity, directional persistence, and cargo sorting efficiency through automated tracking software.

What is known about the structural biology of VPS26B and how does it inform antibody epitope selection?

The structural biology of VPS26B informs strategic epitope selection for antibody development. VPS26B adopts an arrestin-like fold with two lobes connected by a polar core, creating a concave surface that interacts with other retromer components and potentially cargo proteins . This structural information guides epitope selection for antibody generation.

Commercially available VPS26B antibodies target distinct regions of the protein. Some target synthetic peptides derived from amino acids 231-280 of human VPS26B , while others use recombinant full-length protein spanning positions F3-N333 . These different targeting strategies influence antibody performance in various applications.

For structural studies, antibodies targeting surface-exposed loops are preferred for immunoprecipitation applications, as these regions remain accessible in the native protein. Conversely, antibodies recognizing internal epitopes may perform better in applications involving denatured proteins, such as western blotting. Researchers designing custom antibodies should analyze structural data to identify epitopes that are: (1) unique to VPS26B versus VPS26A to ensure specificity, (2) conserved across species if cross-reactivity is desired, and (3) positioned away from functional interfaces if antibodies will be used in functional studies.

Structural data also informs the interpretation of antibody-based experiments. For example, epitopes that become masked upon complex formation could yield false-negative results in co-immunoprecipitation studies. Similarly, antibodies targeting regions involved in cargo binding might interfere with trafficking assays. These structural considerations are essential for selecting optimal antibodies for specific experimental questions about VPS26B function.

How do the applications of monoclonal versus polyclonal VPS26B antibodies differ in research contexts?

The current research landscape for VPS26B antibodies is dominated by polyclonal antibodies, with several commercial options validated for multiple applications . These polyclonal antibodies recognize multiple epitopes on VPS26B, providing high sensitivity but variable batch-to-batch reproducibility. The search results do not specifically mention validated monoclonal antibodies for VPS26B, though this represents an important methodological consideration for researchers.

Monoclonal antibodies, though not specifically mentioned in the search results for VPS26B, would offer different advantages: exceptional specificity for a single epitope, high reproducibility between batches, and suitability for standardized assays. Their monospecificity would be valuable for distinguishing between VPS26A and VPS26B, which share structural similarities as retromer components.

For optimal research strategy, preliminary studies might employ polyclonal antibodies to maximize detection sensitivity, followed by monoclonal antibodies for highly specific applications requiring standardization across experiments. Cross-validation using antibodies recognizing different epitopes provides the most rigorous approach to confirming VPS26B-specific findings.

How does VPS26B function compare across different model organisms used in research?

The function of VPS26B across model organisms shows evolutionary conservation with species-specific adaptations. Available anti-VPS26B antibodies demonstrate reactivity with human, mouse, and rat VPS26B , suggesting structural conservation across mammalian species. This cross-reactivity facilitates comparative studies using the same antibody reagents.

Methodologically, comparative studies across species should begin with sequence alignment and structural prediction to identify conserved domains and species-specific features. Expression analysis across tissues in different organisms can reveal evolutionary shifts in regulation. Functional complementation experiments, where human VPS26B is expressed in model organisms with VPS26B knockouts, can assess functional conservation.

For non-mammalian models, careful validation of antibody cross-reactivity is essential, as epitope conservation may vary. If commercial antibodies fail to recognize orthologues in lower organisms, species-specific antibody generation may be necessary. CRISPR-based genome engineering to tag endogenous VPS26B with conserved epitopes offers an alternative strategy for consistent detection across species.

Phenotypic analysis of VPS26B mutants across species can reveal evolutionarily conserved versus divergent functions, particularly regarding cargo specificity and interaction with different sorting nexin proteins, which have undergone significant diversification during evolution.

What methodological differences exist when studying VPS26B in normal versus disease states?

Studying VPS26B in normal versus disease states requires methodological adaptations across several dimensions. In normal tissues, baseline expression and localization patterns should be established using validated antibodies across multiple techniques . Immunohistochemistry with tissue microarrays allows systematic profiling across organs, while subcellular fractionation followed by western blotting quantifies distribution between membrane compartments. Proximity labeling approaches can map the interactome of VPS26B in healthy cells, establishing a reference for disease comparisons.

In disease contexts, particularly in cancer where VPS26A has demonstrated prognostic relevance , researchers should employ quantitative approaches to assess expression changes. This includes qRT-PCR for transcript levels, western blotting with internal loading controls for protein expression , and digital pathology quantification of immunohistochemistry to assess both expression levels and altered localization patterns. Single-cell analyses provide resolution of heterogeneous expression within diseased tissues.

Functional assessment differs between contexts as well. In normal cells, acute perturbation through siRNA or CRISPR provides insight into homeostatic functions. In disease models, rescue experiments where VPS26B is restored to normal levels are critical for establishing causality. Patient-derived materials present unique challenges, including tissue preservation issues affecting epitope accessibility, requiring optimization of antigen retrieval protocols .

Statistical approaches must account for increased variability in disease samples, often requiring larger sample sizes and paired normal-disease comparisons when possible. Integration of VPS26B data with clinical parameters enables correlation with disease progression and outcome, potentially revealing biomarker applications similar to those established for VPS26A in pancreatic cancer .