snx10b Antibody

What is SNX10 and why is it significant in cellular research?

SNX10 is a 201 amino acid protein that contains a phox (PX) domain and belongs to the sorting nexin family, characterized by phospholipid binding and receptor interaction capabilities. It plays a crucial role in intracellular trafficking and protein sorting, essential processes for maintaining cellular homeostasis and function . SNX10 participates in protein sorting and recycling within endosomes, contributing to membrane protein level regulation and signaling pathways. Its function is vital for cellular processes like receptor internalization and degradation, impacting various physiological responses and disease states . Recent research has identified SNX10 mutations as accounting for approximately 4% of all human autosomal recessive osteopetrosis (ARO), a genetically heterogeneous disorder caused by reduced bone resorption by osteoclasts .

What types of SNX10 antibodies are currently available for research applications?

Researchers can access several types of SNX10 antibodies with varying properties:

• Monoclonal antibodies: Mouse-derived monoclonal antibodies like SNX10 Antibody (1G5), which is an IgG2a isotype antibody that detects SNX10 of human origin , and clone OTI3F1, which reacts with human, mouse, and rat samples .

• Polyclonal antibodies: Rabbit-derived polyclonal antibodies such as 26727-1-AP, which shows reactivity with human, mouse, and rat samples .

• Conjugated variants: Some antibodies are available in conjugated forms, including biotinylated and HRP-conjugated versions for specialized applications .

Each antibody type offers different advantages depending on experimental requirements, with monoclonals providing high specificity and polyclonals offering broader epitope recognition.

What are the validated applications for SNX10 antibodies in research protocols?

SNX10 antibodies have been validated for multiple laboratory applications, with specific dilution recommendations:

The 26727-1-AP antibody has shown positive Western blot detection in mouse and rat brain tissues, while IHC applications have been validated with human pancreas cancer tissue samples . Proper titration in each testing system is recommended to obtain optimal results for specific experimental conditions.

What are the proper storage and handling protocols for SNX10 antibodies?

For optimal antibody performance and longevity, researchers should follow these storage and handling guidelines:

• Store antibodies at -20°C as received .

• Most SNX10 antibodies are provided in PBS buffer containing preservatives such as 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Antibodies are typically stable for one year after shipment when stored properly .

• For the 26727-1-AP antibody, aliquoting is unnecessary for -20°C storage, simplifying laboratory workflow .

• When working with antibodies, minimize freeze-thaw cycles to preserve antibody integrity and activity.

• Some preparations (20μl sizes) may contain 0.1% BSA, which should be considered when designing experiments sensitive to BSA presence .

How should researchers optimize Western blotting protocols for SNX10 detection?

When designing Western blot experiments for SNX10 detection, consider these methodological recommendations:

• Sample preparation: Extract proteins from tissues like brain tissue (mouse/rat) or cell lines expressing SNX10 using standard lysis buffers .

• Gel selection: Use SDS-PAGE gels appropriate for visualizing proteins in the 23-25 kDa range, as SNX10 has a calculated molecular weight of 24 kDa and observed molecular weight of 25 kDa .

• Antibody dilution: Use appropriate dilutions (e.g., 1:1000-1:8000 for 26727-1-AP or 1:2000 for OTI3F1) .

• Controls: Include positive controls like mouse/rat brain tissue which have demonstrated positive signals in validation studies .

• Blocking and incubation: Follow standard blocking procedures using BSA or non-fat milk, and optimize primary antibody incubation times (typically overnight at 4°C).

• Detection: Use appropriate secondary antibodies compatible with the host species (mouse for 1G5 and OTI3F1; rabbit for 26727-1-AP) .

Researchers should expect to observe a band at approximately 25 kDa, although slight variations may occur depending on post-translational modifications or tissue-specific expression patterns.

What considerations should be made when using SNX10 antibodies for immunohistochemistry?

For successful immunohistochemistry studies with SNX10 antibodies:

• Tissue preparation: Use properly fixed and processed tissue specimens. Antibody 26727-1-AP has been validated for human pancreas cancer tissue .

• Antigen retrieval: For the 26727-1-AP antibody, suggested antigen retrieval methods include using TE buffer pH 9.0, or alternatively citrate buffer pH 6.0 .

• Antibody dilution: Use appropriate dilutions (1:50-1:500 for 26727-1-AP) .

• Detection systems: Select detection systems appropriate for the primary antibody host species.

• Controls: Include positive and negative controls to validate staining specificity.

• Counterstaining: Use appropriate counterstains to facilitate visualization of tissue architecture while maintaining visibility of SNX10 staining.

Since SNX10 is involved in intracellular trafficking, expect predominantly cytoplasmic staining patterns with potential enrichment in endosomal compartments.

How can researchers address inconsistent results when using SNX10 antibodies?

When troubleshooting inconsistent results with SNX10 antibodies, consider these methodological adjustments:

• Antibody validation: Verify antibody specificity using known positive controls like brain tissue for Western blotting .

• Sample quality: Ensure protein integrity by using fresh samples and appropriate protease inhibitors during extraction.

• Protocol optimization: Titrate antibody concentrations, as recommended dilutions can vary widely (1:1000-1:8000 for WB, 1:50-1:500 for IHC) .

• Cross-reactivity assessment: Be aware that many SNX10 antibodies react with human, mouse, and rat samples, but specificity may vary between applications .

• Buffer compatibility: Ensure compatibility between sample buffers and antibody storage buffers, particularly when working with high glycerol concentrations (many SNX10 antibodies are supplied in 50% glycerol) .

• Application-specific optimization: Different applications (WB, IHC, IP, ELISA) may require different antibody preparations and dilutions for optimal results.

How can SNX10 antibodies be utilized to investigate osteopetrosis pathophysiology?

Given the established link between SNX10 mutations and autosomal recessive osteopetrosis (ARO) , researchers can employ these methodological approaches:

• Mutation-specific analyses: Use SNX10 antibodies to compare protein expression and localization between wild-type and mutant samples in cellular and tissue models.

• Osteoclast studies: Implement immunofluorescence with SNX10 antibodies to examine protein localization in osteoclasts from normal versus osteopetrotic samples.

• Functional studies: Combine SNX10 antibody-based detection with functional assays measuring bone resorption capacity to correlate SNX10 expression with osteoclast activity.

• Co-localization experiments: Use dual immunostaining with SNX10 antibodies and markers for endosomal compartments to assess trafficking defects in mutant cells.

• Animal model validation: Apply SNX10 antibodies in immunohistochemistry of bone sections from animal models of osteopetrosis to validate disease mechanisms.

These approaches can provide valuable insights into how SNX10 mutations affect osteoclast function and lead to the development of osteopetrosis.

What methodologies enable effective study of SNX10 interactions with other proteins?

To investigate SNX10's interactions with other proteins in the sorting nexin family and beyond:

• Co-immunoprecipitation: Use SNX10 antibodies like 1G5, which has been validated for immunoprecipitation applications , to pull down SNX10 and identify interacting partners through mass spectrometry or Western blotting.

• Proximity labeling: Combine SNX10 antibodies with proximity labeling techniques such as BioID or APEX to identify proteins in close spatial proximity to SNX10 in living cells.

• Sequential immunoprecipitation: Perform sequential IPs with different antibodies to isolate specific SNX10-containing complexes.

• Cross-linking studies: Implement protein cross-linking before immunoprecipitation with SNX10 antibodies to capture transient interactions.

• Fluorescence microscopy: Use SNX10 antibodies in combination with antibodies against potential interacting partners to assess co-localization patterns in fixed cells.

When designing these experiments, researchers should consider the structural features of SNX10, particularly its phox domain, which mediates phospholipid binding and protein interactions characteristic of the sorting nexin family .

How should researchers approach the study of SNX10 in membrane trafficking mechanisms?

To investigate SNX10's role in membrane trafficking:

• Subcellular fractionation: Use SNX10 antibodies to detect the protein in different cellular fractions, particularly endosomal compartments, following subcellular fractionation protocols.

• Live-cell imaging: Combine immunofluorescence using SNX10 antibodies with endosomal markers to track dynamic changes in protein localization.

• Cargo trafficking assays: Use SNX10 antibodies to correlate SNX10 expression or localization with the trafficking of specific cargo molecules through the endosomal system.

• Knockdown/knockout validation: Employ SNX10 antibodies to confirm successful knockdown or knockout in studies investigating the functional consequences of SNX10 depletion on membrane trafficking.

• Endosome morphology analysis: Utilize SNX10 immunostaining to assess changes in endosomal morphology or distribution following experimental manipulations.

These approaches can provide insights into SNX10's role in intracellular trafficking and protein sorting, which are essential processes for maintaining cellular homeostasis and function .

How can researchers accurately quantify SNX10 expression levels from Western blot data?

For rigorous quantification of SNX10 expression:

• Standardization: Use purified recombinant SNX10 protein to generate standard curves for absolute quantification when necessary.

• Loading controls: Normalize SNX10 signals to appropriate loading controls (e.g., β-actin, GAPDH) to account for loading variations.

• Multiple technical replicates: Perform at least three technical replicates to account for blotting and detection variability.

• Exposure optimization: Capture images within the linear range of detection to avoid signal saturation.

• Software analysis: Use specialized software (ImageJ, Image Lab, etc.) for densitometric analysis of band intensity.

• Statistical analysis: Apply appropriate statistical tests to determine significance when comparing expression across different conditions or samples.

When interpreting results, consider that SNX10 has an observed molecular weight of approximately 25 kDa , but variations may occur due to post-translational modifications or experimental conditions.

What explains variations in SNX10 molecular weight observed across different experimental systems?

Researchers frequently observe variations in the apparent molecular weight of SNX10:

• Theoretical vs. observed weight: While the calculated molecular weight of SNX10 is 24 kDa (based on its 201 amino acid sequence), the observed molecular weight is typically around 25 kDa .

• Post-translational modifications: Phosphorylation, ubiquitination, or other modifications can alter protein migration patterns in SDS-PAGE.

• Tissue-specific differences: Expression of different isoforms or tissue-specific post-translational modifications may result in different apparent molecular weights.

• Experimental conditions: Variations in sample preparation, gel percentage, or running conditions can affect protein migration.

• Antibody specificity: Different antibodies may recognize different epitopes or isoforms, resulting in detection of bands at slightly different molecular weights.

To address these variations, researchers should include appropriate positive controls in their experiments and validate findings using multiple antibodies when possible.

How can researchers differentiate between non-specific binding and true SNX10 signal?

To distinguish between specific and non-specific signals:

• Multiple antibodies: Validate findings using different antibodies that recognize distinct epitopes of SNX10 (e.g., monoclonal 1G5 and polyclonal 26727-1-AP ).

• Knockout/knockdown controls: Include samples from SNX10 knockout or knockdown systems as negative controls.

• Peptide competition: Pre-incubate the antibody with purified SNX10 protein or immunizing peptide to block specific binding.

• Tissue/cell specificity: Compare signals across tissues known to express different levels of SNX10 (e.g., brain tissue has shown positive Western blot results ).

• Immunoprecipitation followed by mass spectrometry: Confirm the identity of detected bands through mass spectrometric analysis of immunoprecipitated proteins.

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight of approximately 25 kDa .

These approaches can help ensure the specificity of SNX10 detection in various experimental applications.

What role does SNX10 play in diseases beyond osteopetrosis, and how can antibodies facilitate this research?

While SNX10 mutations are established in osteopetrosis pathophysiology , research into other disease associations can be approached through:

• Expression profiling: Use SNX10 antibodies to compare expression levels across healthy and diseased tissues using tissue microarrays and immunohistochemistry.

• Biomarker potential: Investigate SNX10 as a potential biomarker in diseases associated with altered endosomal trafficking using antibody-based detection methods.

• Genetic correlation studies: Combine genetic analyses of SNX10 variants with antibody-based protein expression studies to identify genotype-phenotype correlations.

• Therapeutic target validation: Use SNX10 antibodies to monitor protein expression or localization changes in response to experimental therapeutics targeting endosomal pathways.

• Cancer research: Given that SNX10 antibodies have been validated in pancreatic cancer tissue , explore potential roles in cancer cell biology, particularly related to receptor recycling and signaling.

These approaches can expand our understanding of SNX10's role in various disease processes beyond its established function in osteopetrosis.

How can SNX10 antibodies be incorporated into high-throughput screening approaches?

Researchers can leverage SNX10 antibodies in high-throughput contexts through:

• Automated immunofluorescence: Implement high-content screening using SNX10 antibodies to assess protein localization changes in response to drug treatment or genetic perturbations.

• ELISA-based screens: Develop quantitative ELISA assays using SNX10 antibodies like 1G5 or 26727-1-AP, which have been validated for ELISA applications .

• Flow cytometry: Adapt SNX10 antibodies for intracellular staining in flow cytometry to quantify expression levels across large cell populations.

• Reverse phase protein arrays: Incorporate SNX10 antibodies into RPPA platforms for screening SNX10 expression across multiple samples simultaneously.

• Multiplexed approaches: Combine SNX10 antibodies with antibodies against other proteins in multiplexed detection systems to assess pathway alterations.

These high-throughput applications can facilitate drug discovery efforts targeting SNX10 or related trafficking pathways and enable large-scale screening of SNX10 expression or localization changes.

What novel functional roles of SNX10 are being uncovered through antibody-based approaches?

Beyond SNX10's established role in endosomal trafficking, antibodies are helping elucidate new functions:

• Endosome homeostasis: Using SNX10 antibodies for localization studies has supported its role in regulating endosome homeostasis .

• Receptor recycling pathways: Immunofluorescence studies using SNX10 antibodies can map its involvement in specific receptor recycling pathways beyond previously characterized interactions.

• Membrane remodeling: Antibody-based detection can help characterize SNX10's role in membrane remodeling processes, particularly through its PX domain interactions with phospholipids .

• Cell-type specific functions: Application of SNX10 antibodies across different cell types is revealing tissue-specific functions beyond its well-characterized role in osteoclasts.

• Signal transduction modulation: Co-immunoprecipitation experiments with SNX10 antibodies are identifying new roles in modulating signal transduction pathways through protein-protein interactions.

These emerging applications are expanding our understanding of SNX10's diverse cellular functions beyond its classical role in intracellular trafficking.