SNX5 Antibody, Biotin conjugated

What is SNX5 and what cellular functions is it involved in?

SNX5 (sorting nexin 5) is a member of the sorting nexin family that interacts with endocytic membranes to regulate vesicular trafficking and macropinocytosis. It plays critical roles in several cellular processes, including antigen presentation in B cells, endolysosomal trafficking, virus-induced autophagy, and immune response regulation . Recent research has shown that SNX5 is essential for actin-dependent plasma membrane remodeling in B cells involved in antigen screening and immune synapse formation . Additionally, SNX5 has been identified as a binding partner of SNX1 and defined as the Vps17p homolog of mammalian cells for the retromer complex, highlighting its importance in intracellular trafficking pathways .

What is the molecular weight and structure of SNX5 protein?

SNX5 is a 404 amino acid protein with a calculated molecular weight of 47 kDa, which corresponds to its observed molecular weight on Western blots . The gene encoding SNX5 has been identified with the ID 27131 in the NCBI database and UniProt ID Q9Y5X3 . SNX5 contains a phospholipid-binding motif (PX domain) that enables it to interact with membrane phospholipids, facilitating its recruitment to endosomal membranes and participation in membrane trafficking events .

What species reactivity has been confirmed for SNX5 antibodies?

Available SNX5 antibodies have demonstrated confirmed reactivity with human, mouse, and rat samples . This cross-species reactivity makes these antibodies valuable tools for comparative studies across different model systems. Validation tests have been performed in multiple cell lines including HEK-293T, K-562, Jurkat, U2OS, and Neuro-2a cells, as well as in mouse and human kidney tissues, confirming the antibody's specificity and utility across diverse experimental systems .

What are the recommended applications for biotin-conjugated SNX5 antibody?

While the search results primarily discuss unconjugated SNX5 antibodies, biotin-conjugated versions would be applicable for similar experimental approaches with additional advantages for signal amplification. Based on validated applications of SNX5 antibodies, a biotin-conjugated version would be suitable for Western blotting (WB), immunohistochemistry (IHC), immunoprecipitation (IP), and ELISA . The biotin conjugation would particularly enhance detection sensitivity in IHC and ELISA applications through avidin/streptavidin-based amplification systems. For microscopy applications, the biotin-conjugated antibody would be valuable for studying SNX5's dynamic localization during processes such as macropinocytosis and antigen presentation .

What dilutions are optimal for different applications of SNX5 antibodies?

Based on experimental validations, the following dilutions are recommended:

• Western Blot (WB): 1:1000-1:4000

• Immunohistochemistry (IHC): 1:100-1:400

• Immunoprecipitation (IP): 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

For biotin-conjugated formats, these dilutions may require optimization, but they provide a starting point. It is advised to titrate the antibody in each testing system to obtain optimal results, as the optimal dilution may be sample-dependent .

How should samples be prepared for SNX5 detection in various applications?

For Western blot analysis of SNX5, standard protein extraction protocols are suitable, with samples typically run on SDS-PAGE gels to detect the 47 kDa band . For immunoprecipitation of endogenous SNX5, successful protocols have been reported using primary macrophages lysed and precipitated with anti-SNX5 antibodies, followed by immunoblotting for interacting partners like SNX1 . For immunohistochemistry, antigen retrieval is recommended with TE buffer at pH 9.0, although citrate buffer at pH 6.0 may also be used as an alternative . When studying SNX5 in the context of virus-induced autophagy, GFP-LC3 co-localization assays have proven effective in visualizing autophagosome formation and SNX5's role in this process .

What controls should be included when using biotin-conjugated SNX5 antibody?

When using biotin-conjugated SNX5 antibody, several controls are essential. Positive controls should include tissues or cell lines with known SNX5 expression such as HEK-293T, Jurkat, or kidney tissues . Negative controls should include SNX5 knockout or knockdown samples; published studies have successfully used CRISPR/Cas9-generated SNX5 KO cells or siRNA-based knockdown approaches . To control for non-specific binding due to the biotin conjugation, a biotin-conjugated isotype control (rabbit IgG) should be used at equivalent concentrations. For validation of specificity, researchers should consider blocking experiments with recombinant SNX5 protein to confirm signal reduction.

What are common issues in SNX5 detection and how can they be resolved?

Common issues in SNX5 detection include insufficient signal strength, non-specific binding, and inconsistent results across different sample types. For weak signals in Western blots, researchers should optimize protein loading (30-50 μg is typically sufficient), reduce dilution of primary antibody, or extend incubation time. Non-specific binding can be addressed by increasing blocking time or concentration of blocking agent, and using more stringent washing conditions. If inconsistent results are observed between different samples, standardizing sample preparation protocols and ensuring equal protein loading are essential steps. For biotin-conjugated antibodies specifically, high background can result from endogenous biotin; this can be addressed using a biotin blocking kit prior to antibody application.

How can specificity of SNX5 antibody binding be confirmed?

Antibody specificity can be confirmed through multiple approaches. The gold standard is showing reduced or absent signal in SNX5 knockout or knockdown samples. Published studies have successfully used CRISPR/Cas9-generated SNX5 KO cells and siRNA-based approaches targeting SNX5 . For example, SNX5 miRNA-1.4 adenovirus has been shown to reduce SNX5 levels by ~92% compared to control miRNA . Another validation approach is reconstitution experiments, where introducing wild-type SNX5 into knockout cells should restore the signal, as demonstrated in two independent SNX5 KO/GFP-LC3/SNX5 cell clones . Additionally, peptide competition assays using the immunogen (SNX5 fusion protein Ag12330) can confirm binding specificity .

How should contradictory results in SNX5 functional studies be interpreted?

Contradictory results in SNX5 functional studies may arise from several factors that require careful consideration. First, SNX5's functions appear to be cell-type specific; for instance, its role in macropinocytosis is prominent in macrophages but may differ in other cell types . Second, functional redundancy with related proteins like SNX32 should be considered, as knockdown of both SNX5 and SNX32 has shown similar effects on autophagy induction . Third, the experimental context matters significantly - SNX5's role in virus-induced autophagy differs from its function in basal or starvation-induced autophagy . When encountering contradictory results, researchers should carefully compare experimental conditions, cell types, knockdown efficiency, and whether related proteins might compensate for SNX5 deficiency.

How can biotin-conjugated SNX5 antibody be used to study macropinocytosis?

Biotin-conjugated SNX5 antibody can be employed in several approaches to study macropinocytosis. For fixed-cell imaging, it can be used in conjunction with streptavidin-conjugated fluorophores to visualize SNX5 localization during macropinosome formation. Research has shown that SNX5 is essential for efficient macropinocytosis and antigen processing in primary macrophages . Quantitative analysis of macropinocytosis can be performed by combining SNX5 immunostaining with dextran uptake assays, as previous studies have demonstrated that depletion of SNX5 results in dramatic reduction in uptake and processing of soluble antigens in macrophages . To connect SNX5 localization with functional outcomes, researchers can combine SNX5 immunostaining with antigen processing assays, tracking the internalization and processing of labeled antigens in the presence or absence of SNX5.

What approaches can be used to study SNX5's role in virus-induced autophagy?

To investigate SNX5's role in virus-induced autophagy, researchers can employ several sophisticated approaches. Co-localization studies using biotin-conjugated SNX5 antibody and markers of autophagy (such as LC3) can reveal spatial relationships during viral infection. Genome-wide siRNA screens have identified SNX5 as essential for virus-induced, but not basal or starvation-induced autophagy . Viral infectivity and replication assays in SNX5 knockout or knockdown cells have demonstrated increased susceptibility to multiple viruses, including Sindbis virus, HSV-1, West Nile virus, and chikungunya virus . To establish causality, reconstitution experiments with wild-type SNX5 in knockout cells should restore antiviral autophagy. For in vivo relevance, mouse models with SNX5 deficiency (Snx5-/- mice) have shown increased susceptibility to lethal viral infections, providing a system to study the impact of SNX5 on viral pathogenesis .

How can SNX5 antibodies help investigate protein-protein interactions?

SNX5 antibodies are valuable tools for investigating protein-protein interactions through several methods. Immunoprecipitation experiments using SNX5 antibodies have successfully demonstrated interactions between endogenous SNX5 and SNX1 in primary macrophages . Co-immunoprecipitation followed by mass spectrometry can identify novel SNX5 binding partners beyond known interactions. Proximity ligation assays (PLA) using biotin-conjugated SNX5 antibody and antibodies against suspected interaction partners can visualize interactions in situ with high sensitivity. Research has shown that SNX5 interacts with autophagy-specific PI3KC3-C1 complexes (containing ATG14) but not PI3KC3-C2 complexes (containing UVRAG) , demonstrating the specificity of its protein interactions. For studying dynamics of interactions, live-cell imaging techniques combining fluorescently labeled interacting partners with SNX5 can reveal temporal aspects of these associations.

How should SNX5 expression data be normalized across different experimental conditions?

When analyzing SNX5 expression data, proper normalization is crucial for valid comparisons across different experimental conditions. For Western blot quantification, normalization to housekeeping proteins (β-actin, GAPDH, or tubulin) is standard practice. When comparing SNX5 expression in different tissues or cell types, researchers should be aware that baseline expression levels may vary naturally; thus, relative changes within each system may be more informative than absolute comparisons. For mRNA expression analysis, qPCR data should be normalized to stable reference genes that are not affected by the experimental conditions. When analyzing SNX5 localization changes rather than expression levels, quantification should focus on the percentage of SNX5 in specific cellular compartments relative to total cellular SNX5, rather than absolute fluorescence intensity.

What statistical approaches are appropriate for analyzing SNX5 knockdown experiments?

For analyzing SNX5 knockdown experiments, statistical approaches should be selected based on experimental design and data distribution. For comparing two conditions (e.g., control vs. knockdown), Student's t-test is appropriate if data follows normal distribution; otherwise, non-parametric tests like Mann-Whitney U test should be used. For multiple experimental groups, ANOVA followed by appropriate post-hoc tests (e.g., Tukey's or Dunnett's) is recommended. When analyzing time-course experiments examining SNX5's role in dynamic processes like autophagy or macropinocytosis, repeated measures ANOVA or mixed-effects models may be more appropriate. Power analysis should be performed to determine adequate sample sizes, particularly for in vivo experiments with SNX5 knockout mice, where a minimum of 8-10 animals per group is typically needed for survival studies .

How can changes in SNX5 localization be quantified in microscopy images?

Quantification of SNX5 localization changes in microscopy images requires rigorous image analysis approaches. For co-localization analysis, Pearson's or Mander's correlation coefficients can measure the degree of overlap between SNX5 and markers of specific cellular compartments. Line scan analysis across cellular regions can provide quantitative profiles of SNX5 distribution relative to membrane or organelle markers. For dynamic processes like immune synapse formation, where SNX5 accumulates at specific sites, measuring the percentage of SNX5 signal at the synapse relative to total cellular SNX5 can provide meaningful quantification . When studying SNX5's role in macropinosome formation, tracking the enrichment of SNX5 at membrane ruffles and nascent macropinosomes over time can reveal its temporal recruitment patterns . For all quantitative imaging approaches, automated image analysis pipelines using software like ImageJ/Fiji, CellProfiler, or custom scripts in Python or MATLAB are recommended to reduce bias and increase throughput.