VPS28 Antibody, Biotin conjugated

What is VPS28 and what cellular processes does it regulate?

VPS28 (Vacuolar Protein Sorting-Associated Protein 28) is a critical component of the ESCRT-I complex that regulates vesicular trafficking processes within cells. It is primarily localized to the cell membrane and plays essential roles in endosomal sorting and multivesicular body formation. VPS28 has a gene ID of 51160 and Swiss Prot accession number Q9UK41, with synonyms including H-Vps28 and ESCRT-I complex subunit VPS28 . The protein is involved in the precise trafficking of cellular components, making it crucial for maintaining cellular homeostasis. Research indicates that VPS28 dysregulation may contribute to pathological conditions, particularly in cancer progression, where alterations in vesicular trafficking can impact cell signaling and protein degradation pathways.

Why choose a biotin-conjugated VPS28 antibody over non-conjugated alternatives?

Biotin-conjugated VPS28 antibodies offer significant advantages for immunodetection techniques due to biotin's exceptionally high affinity for avidin and streptavidin. This interaction provides a robust platform for signal amplification, enhancing detection sensitivity compared to direct labeling methods . The biotin-streptavidin system allows for flexible experimental design where the primary antibody (anti-VPS28) can be visualized using various streptavidin-conjugated reporter molecules (fluorophores, enzymes). Additionally, biotin conjugation with spacers (Biotin-SP) positions the biotin moiety away from the antibody surface, improving accessibility to streptavidin binding sites and further enhancing detection sensitivity . This flexibility is particularly valuable when studying proteins like VPS28 that may be expressed at relatively low levels in certain cell types or when conducting multiplexed detection experiments.

What are the typical applications for VPS28 antibodies in research?

VPS28 antibodies have demonstrated efficacy across multiple immunological applications. Based on validated data from antibody providers, the most common applications include:

These applications enable researchers to detect, localize, and quantify VPS28 expression across various experimental contexts, from protein lysates to tissue sections and cultured cells . The versatility of VPS28 antibodies makes them valuable tools for investigating this protein's role in both normal cellular functions and disease states.

How should I validate a VPS28 biotin-conjugated antibody before use in critical experiments?

Comprehensive validation of a VPS28 biotin-conjugated antibody requires multiple approaches to ensure specificity and reliability. Begin with positive and negative control tissues or cell lines with known VPS28 expression levels. Based on published research, T47D and MCF7 breast cancer cell lines express high levels of VPS28 and can serve as positive controls . For negative controls, consider using VPS28 knockdown models created through shRNA technology as described in recent publications .

Western blotting validation should confirm a single band at the expected molecular weight (25 kDa) in multiple cell types, including HepG2, Jurkat, A549, and human fetal brain and liver tissues . For immunohistochemistry or immunofluorescence validation, compare staining patterns with published literature and verify subcellular localization (primarily cell membrane) . Additionally, perform peptide competition assays where pre-incubation with the immunizing peptide should abolish specific staining. For biotin conjugates specifically, include streptavidin-only controls to identify any non-specific binding due to endogenous biotin in your experimental system.

What are the optimal storage conditions and shelf-life considerations for biotin-conjugated VPS28 antibodies?

Biotin-conjugated VPS28 antibodies require specific storage conditions to maintain their immunoreactivity and conjugate stability. According to manufacturer recommendations, these antibodies should be stored at -20°C for optimal long-term stability, with an expected shelf-life of 12 months under these conditions . The antibodies are typically supplied in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.02% Proclin300, and 50% Glycerol, which helps prevent freeze-thaw damage .

To maximize antibody performance and longevity, minimize freeze-thaw cycles by aliquoting the antibody upon receipt. Evidence suggests that biotin conjugates are particularly sensitive to repeated freeze-thaw cycles, which can compromise both antibody binding and biotin activity. When working with the antibody, always keep it on ice or at 4°C and return to -20°C promptly after use. Monitor for signs of precipitation or changes in performance, which may indicate degradation. Some manufacturers offer biotin conjugations made fresh to order with delivery lead times of up to 3 weeks, suggesting the benefits of working with freshly conjugated antibodies for optimal performance .

How do I optimize the signal-to-noise ratio when using biotin-conjugated VPS28 antibodies in immunohistochemistry?

Optimizing signal-to-noise ratio with biotin-conjugated VPS28 antibodies requires systematic troubleshooting of several experimental variables. Begin with antibody titration experiments to determine the minimal concentration providing maximum specific signal. Published protocols suggest starting dilutions of 1:200-400 for IHC-P and 1:100-500 for IHC-F applications , but optimal dilutions should be determined empirically for each experimental system.

Effective blocking is critical with biotin-conjugated antibodies. Use avidin/biotin blocking kits before antibody incubation to minimize endogenous biotin interference, particularly in biotin-rich tissues like liver, kidney, and brain. Heat-mediated antigen retrieval with citrate buffer pH 6 has been validated for VPS28 detection in paraffin-embedded tissues . When using streptavidin detection systems, include a streptavidin-only control to identify non-specific binding.

Consider using amplification systems like ABC (Avidin-Biotin Complex) or TSA (Tyramide Signal Amplification) for detecting low-abundance VPS28, but be aware that over-amplification can increase background. For multi-color immunofluorescence, use streptavidin conjugated to spectrally distinct fluorophores and perform sequential detection if using multiple biotin-conjugated primary antibodies. Remember that longer spacers between biotin and antibody (Biotin-SP) may improve detection sensitivity, particularly with alkaline phosphatase-conjugated streptavidin .

What is the current evidence regarding VPS28's role in cancer progression?

Recent research has revealed significant associations between VPS28 expression and cancer progression, particularly in breast cancer. Analysis of The Cancer Genome Atlas (TCGA) and Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases has demonstrated elevated VPS28 expression in breast cancer tissues compared to normal controls, with this overexpression correlating with poor prognosis (p < 0.001) . Survival analysis showed that higher VPS28 expression indicates significantly shorter survival duration (HR = 2.43; 95% CI: 1.44–4.1; p < 0.001) .

At the cellular level, comparative analysis of VPS28 expression across multiple breast cancer cell lines (MDAMB231, BT474, MCF7, T47D, and MDAMB436) versus normal mammary epithelial cells (MCF10A) revealed that T47D and MCF7 cells exhibited the highest VPS28 expression levels, making them valuable models for studying VPS28 function . Functional studies using shRNA-mediated VPS28 knockdown in these high-expressing cell lines demonstrated that VPS28 suppression significantly reduced cell viability, proliferation, migration, and invasion capacities while increasing apoptosis . These findings strongly suggest that VPS28 plays an oncogenic role in breast cancer and may represent a potential therapeutic target or prognostic marker.

How can VPS28 antibodies be used in studies of vesicular trafficking and the ESCRT complex?

VPS28 antibodies serve as crucial tools for investigating the dynamics and regulation of the ESCRT-I complex and broader vesicular trafficking processes. Immunofluorescence studies using VPS28 antibodies (typically at 1:100 dilution) can visualize subcellular localization and potential colocalization with other ESCRT components or cargo proteins . This approach enables researchers to map the spatial-temporal dynamics of ESCRT-mediated sorting in living cells.

For studying protein-protein interactions within the ESCRT-I complex, VPS28 antibodies can be employed in co-immunoprecipitation assays to identify novel binding partners or confirm known interactions. Additionally, chromatin immunoprecipitation (ChIP) experiments can reveal transcriptional regulators of VPS28, complementing studies that have identified microRNA regulators such as miR-491-5p .

In disease models, particularly cancer, VPS28 antibodies provide a means to assess how alterations in vesicular trafficking contribute to pathogenesis. By combining VPS28 immunodetection with markers of endosomal compartments, researchers can investigate whether cancer-associated VPS28 overexpression disrupts normal vesicular trafficking pathways, potentially affecting receptor recycling or degradation to promote sustained growth signaling.

What methodological considerations are important when using VPS28 antibodies for knockout/knockdown validation studies?

When using VPS28 antibodies to validate gene editing or expression knockdown, several methodological considerations are critical for generating reliable results. First, select appropriate antibody applications based on your experimental system. Western blotting offers quantitative assessment of protein reduction at the expected molecular weight (25 kDa) , while immunofluorescence provides insights into changes in subcellular localization and expression heterogeneity across cell populations.

Based on published protocols, VPS28 knockdown models have been successfully generated using short hairpin RNA (shRNA, 1 μg) with lipofectamine-based transfection . When validating VPS28 knockdown by Western blot, research indicates that primary antibodies against VPS28 at 1/1,000 dilution with HRP-labeled secondary antibodies at 1/2,000 dilution produce reliable results . For normalization, β-actin (1/200 dilution) has been validated as an appropriate loading control .

Multiple detection methods should be employed for thorough validation. In addition to protein-level verification by Western blot and immunofluorescence, RT-qPCR confirmation of mRNA reduction provides complementary evidence of successful knockdown. When evaluating functional consequences of VPS28 depletion, published data indicate that proliferation (via MTT assay), migration (via transwell assay), and apoptosis (via flow cytometry) represent the most informative endpoints for phenotypic confirmation .

How can I resolve non-specific binding issues with biotin-conjugated VPS28 antibodies?

Non-specific binding is a common challenge with biotin-conjugated antibodies due to endogenous biotin and potential cross-reactivity. A systematic troubleshooting approach includes several key steps. First, implement a dedicated avidin/biotin blocking step before primary antibody incubation, particularly crucial for biotin-rich tissues like liver, kidney, and brain. If using paraffin-embedded sections, ensure complete deparaffinization and optimize antigen retrieval conditions—heat-mediated retrieval with citrate buffer pH 6 has been validated for VPS28 detection .

Optimize blocking conditions by testing different blocking agents (BSA, normal serum, commercial blockers) at varying concentrations and incubation times. When performing the primary antibody incubation, increase washing stringency by using PBS-T with higher detergent concentrations (0.1-0.3% Tween-20) and extending wash durations. Consider using secondary detection reagents pre-adsorbed against tissue species to minimize cross-reactivity.

For multiplex staining, implement sequential detection protocols and include appropriate controls: primary antibody omission, isotype controls, and streptavidin-only controls. If non-specific nuclear staining persists, add nuclease treatment during sample preparation. Additionally, titrating both primary antibody and streptavidin-conjugated detection reagents can significantly improve signal-to-noise ratio while maintaining specific VPS28 detection.

What are the best approaches for multiplexed detection involving VPS28 biotin-conjugated antibodies?

Successful multiplexed detection with VPS28 biotin-conjugated antibodies requires careful experimental design to prevent cross-reactivity and signal overlap. Sequential detection represents the most reliable approach when one or more biotin-conjugated antibodies are involved. In this method, complete the entire detection process for the biotin-conjugated VPS28 antibody (including streptavidin application and thorough washing) before introducing subsequent primary antibodies.

When designing multiplexed panels, consider using directly labeled primary antibodies for additional targets to avoid multiple biotin-streptavidin interactions. If using multiple biotin-conjugated primaries is unavoidable, employ streptavidin conjugated to spectrally distinct fluorophores with minimal overlap and include appropriate spectral compensation controls.

Tyramide signal amplification (TSA) systems combined with biotin-conjugated antibodies can enable sequential multi-color immunohistochemistry by permanently depositing fluorophores, allowing antibody stripping between rounds while preserving signal. When analyzing cellular components in relation to VPS28, published protocols suggest focusing on cell membrane localization as VPS28's primary subcellular location . For co-localization studies of VPS28 with other ESCRT components, confocal microscopy with careful attenuation of bleed-through is essential for reliable interpretation.

How should I interpret discrepancies in VPS28 detection between different antibody clones or detection methods?

Discrepancies in VPS28 detection across different methodologies require systematic investigation and can reveal important biological insights. First, consider the epitopes targeted by different antibody clones—some VPS28 antibodies are generated against synthetic peptides derived from human VPS28 , while others may target different regions. Epitope accessibility varies between applications; denatured Western blot samples expose different epitopes than fixed tissues or cells in immunohistochemistry.

Post-translational modifications may affect antibody binding. While VPS28's primary reported subcellular location is the cell membrane , modifications might alter localization or epitope accessibility. Verify results using antibodies from different suppliers targeting distinct epitopes. The literature indicates that monoclonal antibodies like EPR9881(B) and polyclonal antibodies like 15478-1-AP have been validated for VPS28 detection, providing complementary approaches.

When analyzing breast cancer samples, contextual interpretation is crucial. Published studies demonstrate that VPS28 overexpression correlates with poor prognosis in breast cancer patients , but expression patterns may vary by cancer subtype. If discrepancies persist between protein and mRNA levels, consider investigating miRNA regulation, as miR-491-5p has been identified as a regulator of VPS28 . These multifaceted approaches can transform apparent technical discrepancies into valuable biological insights about VPS28 regulation and function.

What are the emerging applications of VPS28 antibodies in translational research?

VPS28 antibodies are increasingly valuable in translational research applications connecting basic ESCRT biology to clinical outcomes. Based on recent studies demonstrating VPS28 overexpression in breast cancer and its correlation with poor prognosis (HR = 2.43; 95% CI: 1.44–4.1; p < 0.001) , VPS28 immunodetection holds promise as a potential prognostic biomarker. Immunohistochemical evaluation of VPS28 in tumor biopsies could potentially stratify patients by risk level and inform treatment decisions.

The established role of VPS28 in vesicular trafficking makes its detection relevant in research on therapeutic resistance mechanisms. Many targeted therapies depend on receptor internalization and degradation—processes regulated by the ESCRT machinery. Monitoring VPS28 expression and localization in treatment-resistant versus sensitive tumors may reveal alterations in trafficking pathways contributing to resistance.

As liquid biopsy approaches evolve, VPS28 antibodies may find application in detecting VPS28-positive extracellular vesicles (EVs) in patient samples. Since ESCRT components are involved in EV biogenesis, characterizing VPS28-associated EVs could provide novel biomarkers or insight into cancer cell communication with the tumor microenvironment. These translational applications bridge fundamental research on vesicular trafficking to potential clinical applications in diagnosis, prognosis, and therapeutic monitoring.

How can researchers contribute to improving antibody validation standards for VPS28 detection?

Researchers can substantially advance VPS28 antibody validation by implementing comprehensive, multi-approach validation strategies and sharing detailed methodological information. The ideal validation protocol should include genetic controls (VPS28 knockout or knockdown models) , multiple detection methods (Western blot, immunohistochemistry, immunofluorescence) , and cross-validation with antibodies targeting different VPS28 epitopes.

Documentation and data sharing represent crucial contributions to validation standards. Researchers should thoroughly document all experimental conditions, including sample preparation, antibody concentrations, incubation times, detection systems, and image acquisition parameters. Sharing both positive and negative results, including optimization challenges, through publications, repositories, or resources like Antibodypedia provides invaluable information for the research community.