VPS26A Antibody

What is VPS26A and what cellular functions does it perform?

VPS26A (vacuolar protein sorting 26 homolog A) is one of two paralogs of yeast Vps26 found in mammals (the other being VPS26B) . It functions as a core component of the retromer complex, which consists of VPS26A (or VPS26B), VPS29, VPS35, SNX1, and SNX2 . The retromer complex plays a crucial role in endosomal protein sorting and recycling of transmembrane receptors from endosomes to the trans-Golgi network .

VPS26A specifically defines distinct retromer complexes compared to those containing VPS26B . Research has shown that VPS26A has been linked to several important cellular processes:

• Receptor recycling and trafficking

• Maintenance of cellular homeostasis

• Potentially implicated in neurodegenerative diseases and cancer pathways

The protein has a molecular weight of approximately 38 kDa and is primarily localized to endosomal compartments, as demonstrated by immunofluorescence studies showing distinct endosomal patterns .

How do I select the appropriate VPS26A antibody for my research?

Selection of the optimal VPS26A antibody depends on several methodological considerations:

• Target species reactivity: Available antibodies show different cross-reactivity profiles:

  • Human, mouse, rat, and monkey reactivity (Cell Signaling #99384)

  • Human, mouse, and rat reactivity (Proteintech #12804-1-AP)

  • Human-specific (Abnova MAB15737)

  • Plant-specific (Arabidopsis thaliana) (Agrisera AS13 2632)

• Antibody type (monoclonal vs. polyclonal):

  • Monoclonal antibodies (Cell Signaling #99384, Abnova MAB15737) offer higher specificity and batch-to-batch consistency

  • Polyclonal antibodies (Proteintech #12804-1-AP, Agrisera AS13 2632) typically provide broader epitope recognition

• Intended application:

  • Western blot (all antibodies are validated)

  • Immunohistochemistry (validated for Proteintech, Abnova products)

  • Immunofluorescence (validated for Proteintech, Abnova products)

  • Cell-type specific validation (tissue-specific validation may influence your choice)

• Immunogen information: Consider the epitope region

  • Cell Signaling antibody targets residues surrounding Pro221 of human VPS26A

  • Abnova antibody's immunogen sequence: LRKQRTNFHQRFESPESQASAEQPEM

Always validate antibodies in your specific experimental context before proceeding with larger studies.

What are the validated applications for VPS26A antibodies?

Based on the search results, VPS26A antibodies have been validated for multiple applications. Below is a comprehensive table summarizing the applications and recommended dilutions for different commercially available antibodies:

Several publications have specifically used VPS26A antibodies in various applications, including knockout/knockdown validation studies . When performing these applications, it's recommended to follow the manufacturer's provided protocols for optimal results.

How should I optimize Western blot protocols for VPS26A detection?

Optimizing Western blot protocols for VPS26A detection requires careful consideration of several parameters:

• Sample preparation:

  • VPS26A (38 kDa) has been successfully detected in various cell and tissue lysates including HEK-293 cells, mouse/rat kidney tissues , U-251 MG cells , and Arabidopsis protoplasts .

  • Standard lysis buffers are generally effective, as demonstrated by Agrisera's protocol using buffer containing 100 mM Tris (pH 7.8), 200 mM NaCl, 1 mM EDTA, 2% (v/v) beta-mercaptoethanol, and 0.2% (v/v) Triton X-100 .

• Gel percentage and transfer conditions:

  • A 10-12% SDS-PAGE gel is optimal for resolving the 38 kDa VPS26A protein.

  • Standard wet or semi-dry transfer systems are suitable with PVDF or nitrocellulose membranes.

• Blocking and antibody incubation:

  • For Proteintech antibody: Follow manufacturer's WB protocol optimized for VPS26A .

  • For Cell Signaling antibody: 1:1000 dilution is recommended .

  • For Abnova antibody: 1:200-1:500 dilution range is recommended .

  • For Agrisera antibody: 1:5000 dilution with plant samples .

• Expected results and troubleshooting:

  • The expected band should appear at approximately 38 kDa.

  • Some non-specific bands may appear with certain antibodies; one reviewer noted: "It is Ok to detect endogenous VPS26A although there are some non-specific bands" .

  • If signal is weak, longer exposure times or higher antibody concentrations may be required.

  • Always include positive controls from validated samples (e.g., HEK-293 cells for mammalian studies).

• Loading controls:

  • Standard housekeeping proteins (β-actin, GAPDH, tubulin) can be used as loading controls.

What are the best practices for immunohistochemistry using VPS26A antibodies?

For optimal immunohistochemistry (IHC) results with VPS26A antibodies, consider these methodological guidelines:

• Tissue preparation and antigen retrieval:

  • Fixed tissues: Both formalin-fixed paraffin-embedded (FFPE) tissues have been successfully used .

  • Antigen retrieval: For Proteintech antibody, TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 can alternatively be used .

• Antibody selection and dilution:

  • Proteintech (12804-1-AP): Use at 1:50-1:500 dilution .

  • Abnova (MAB15737): Use at 1:200-1:500 dilution .

  • Positive tissue controls: Human breast cancer tissue, human cervical cancer tissue, and human kidney tissue have been validated for Proteintech antibody . Human fallopian tube has been validated for Abnova antibody .

• Detection systems:

  • Standard HRP/DAB detection systems are compatible.

  • For fluorescent detection, select secondary antibodies appropriate for your primary antibody host species (rabbit for Proteintech, mouse for Abnova).

• Expected staining pattern:

  • VPS26A typically shows cytoplasmic staining with an endosomal pattern.

  • The Abnova antibody shows "strong granular cytoplasmic immunoreactivity in glandular epithelium cells" in human fallopian tube samples .

• Controls and validation:

  • Include positive control tissues known to express VPS26A.

  • Consider including negative controls (omitting primary antibody) and isotype controls.

How can I optimize immunofluorescence protocols for VPS26A detection?

For successful immunofluorescence (IF) detection of VPS26A, follow these methodological guidelines:

• Cell preparation and fixation:

  • Validated cell lines include HeLa cells and MCF7 cells .

  • From customer review data: "Oc-2 cells fixed with 4% PFA Perm. by 0.3% tx100 for 5 min blocked with 1% BSA in 1XPBS for 2 hours" .

• Antibody dilution and incubation:

  • Proteintech (12804-1-AP): Use at 1:50-1:500 dilution .

  • Abnova (MAB15737): Use at 1-4 μg/mL .

  • Customer protocol example: "vps26a antibody incubated 1:300 overnight at 4 degrees in 1%BSA in 1x PBS" .

• Co-staining recommendations:

  • VPS26A antibodies can be effectively combined with markers for:

    • Nuclear staining (DAPI)

    • Cytoskeletal elements (phalloidin for F-actin)

    • Endosomal markers (to confirm co-localization)

  • Example from customer review: "Co-stained with DAPI (blue) and Ph647 (red) to visualize DNA and F-actin respectively" .

• Expected staining pattern:

  • VPS26A typically shows distinct endosomal staining patterns.

  • The Abnova antibody in MCF7 cells "shows distinct endosomes" .

• Image acquisition parameters:

  • Confocal microscopy is recommended for detailed visualization of endosomal structures.

  • Use appropriate exposure settings to avoid bleaching while maintaining signal detection.

How can I validate the specificity of VPS26A antibodies in knockout/knockdown experiments?

Validating antibody specificity through knockout/knockdown approaches is critical for ensuring reliable experimental results. For VPS26A antibodies, consider these methodological approaches:

• Knockout/knockdown validation:

  • According to Proteintech, their VPS26A antibody (12804-1-AP) has been used in at least one published knockout/knockdown validation study .

  • Design siRNA or CRISPR-Cas9 targeting VPS26A to generate knockdown or knockout cell lines.

  • Compare antibody signal between wild-type and KO/KD samples via Western blot, with expected complete loss or significant reduction of the 38 kDa band.

• Rescue experiments:

  • For comprehensive validation, reintroduce VPS26A expression in knockout cells.

  • Observe restoration of antibody signal, confirming specificity.

• Overexpression controls:

  • Complementary to KO/KD approaches, overexpress tagged VPS26A constructs.

  • Verify co-localization of antibody signal with tag-specific antibodies.

  • Check for increased signal intensity in Western blot.

• Tissue-specific considerations:

  • Different antibodies may show variable specificity in different tissues.

  • The Proteintech antibody has been validated in mouse/rat kidney tissues and human breast/cervical cancer tissues .

  • The Agrisera antibody shows reactivity in Arabidopsis but not in Nicotiana tabacum .

• Cross-reactivity assessment:

  • Test for potential cross-reactivity with VPS26B, which shares sequence homology with VPS26A.

  • Compare staining patterns with known VPS26A and VPS26B localization data.

What is the relationship between VPS26A and disease states, and how can antibodies help elucidate these connections?

VPS26A, as a core retromer component, has been implicated in several disease processes. Antibody-based research can help elucidate these connections through the following methodological approaches:

• Cancer research applications:

  • VPS26A antibodies have been used to study pancreatic adenocarcinoma, as indicated by the publication "The Prognostic Value and the Oncological and Immunological Roles of Vacuolar Protein Sorting Associated Protein 26A in Pancreatic Adenocarcinoma" .

  • Methodology: Use IHC to assess VPS26A expression levels in tumor vs. normal tissues (validated in human breast and cervical cancer tissues ).

  • Analyze correlation between expression levels and clinical outcomes.

• Neurodegenerative disease research:

  • VPS26A antibodies have supported research in Down syndrome and Parkinson's disease models:

    • "Retromer proteins reduced in Down syndrome and the Dp16 model: impact of APP dose and preclinical studies of a γ-secretase modulator"

    • "Phospholipase PLA2G6, a Parkinsonism-Associated Gene, Affects Vps26 and Vps35, Retromer Function, and Ceramide Levels, Similar to α-Synuclein Gain"

  • Methodology: Western blot analysis of brain tissue samples to quantify VPS26A levels in disease vs. control samples.

• Metabolic regulation:

  • Research indicates VPS26A involvement in cellular energy homeostasis: "RAB21 controls autophagy and cellular energy homeostasis by regulating retromer-mediated recycling of SLC2A1/GLUT1" .

  • Methodology: Combine IF with glucose transporter antibodies to assess co-localization and trafficking patterns.

• Autophagy connections:

  • VPS26A antibodies have contributed to understanding autophagy mechanisms: "Noncanonical roles of ATG5 and membrane atg8ylation in retromer assembly and function" .

  • Methodology: Use IF to study co-localization with autophagy markers, and Western blot to assess expression changes during autophagy induction.

• Experimental considerations:

  • For disease-related studies, careful sample selection and appropriate controls are critical.

  • Combining antibody-based techniques with functional assays provides more comprehensive understanding.

How can researchers interpret conflicting results between different VPS26A antibodies?

When facing discrepancies in results obtained with different VPS26A antibodies, researchers should consider these methodological approaches for resolution:

What are the emerging applications of VPS26A antibodies in retromer complex and autophagy research?

VPS26A antibodies are increasingly valuable tools in studying retromer biology and its connections to autophagy pathways. Consider these methodological approaches for cutting-edge applications:

• Retromer assembly and function analysis:

  • Publications utilizing VPS26A antibodies have revealed "Noncanonical roles of ATG5 and membrane atg8ylation in retromer assembly and function" .

  • Methodology: Use immunoprecipitation with VPS26A antibodies to pull down retromer complexes under different experimental conditions.

  • Combine with mass spectrometry to identify novel interacting partners.

• Subcellular trafficking studies:

  • VPS26A antibodies enable tracking of retromer-mediated recycling pathways.

  • Referenced research shows connections to glucose transporter trafficking: "RAB21 controls autophagy and cellular energy homeostasis by regulating retromer-mediated recycling of SLC2A1/GLUT1" .

  • Methodology: Combine live-cell imaging with fixed-cell IF to track dynamic retromer-dependent trafficking events.

• Autophagy-retromer crosstalk investigation:

  • Multiple publications indicate connections between VPS26A/retromer and autophagy mechanisms .

  • Methodology:

    • Use IF to assess co-localization between VPS26A and autophagy markers under basal and induced autophagy conditions.

    • Employ Western blot to quantify changes in VPS26A levels during autophagy modulation.

    • Implement proximity ligation assays to detect direct interactions between retromer and autophagy machinery components.

• Advanced imaging applications:

  • Super-resolution microscopy techniques can reveal precise subcellular localization.

  • Methodology:

    • Use STORM or STED microscopy with VPS26A antibodies to visualize endosomal microdomains.

    • Implement live-cell FRAP experiments with fluorescently-tagged VPS26A to study dynamic retromer assembly/disassembly.

• Systems biology approaches:

  • Integration of antibody-based studies with -omics data.

  • Methodology:

    • Combine phosphoproteomics with VPS26A immunoprecipitation to identify regulatory modifications.

    • Correlate VPS26A expression/localization data with transcriptomic profiles under various cellular stresses.