SNX14 Antibody, HRP conjugated

What is SNX14 and what are its main structural features?

SNX14 is a member of the sorting nexin family of proteins that contains multiple functional domains including a regulator of G-protein signaling (RGS) domain, a PX (phox homology) domain, an N-terminal hydrophobic region, and a PXA (PX-associated) domain. The PXA domain is specifically responsible for binding to organelle membranes, while the PX domain mediates interactions with phosphoinositides. SNX14 is an ER-anchored integral membrane protein involved in cellular lipid transport . The protein's structural organization enables its diverse functions in endosomal trafficking and cell signaling pathways.

Where is SNX14 predominantly expressed in human tissues?

SNX14 exhibits tissue-specific expression patterns with particularly high levels in the brain, including the hippocampus, nucleus accumbens, and cerebellum. It is also abundantly expressed in lung and testis tissues . Within the central nervous system, SNX14 is detected in primary hippocampal neurons, glial cells, and hippocampal cell lines like HT-22. Interestingly, SNX14 shows a mutually exclusive tissue distribution pattern compared to SNX13, which is abundant in heart and muscle tissues where SNX14 is virtually absent . This specific expression pattern suggests specialized functions for SNX14 in neuronal tissues.

What are the main applications of SNX14 antibodies in research?

SNX14 antibodies, particularly HRP-conjugated versions, serve multiple research purposes including:

• Protein detection via Western blotting to assess expression levels

• Immunohistochemistry and immunofluorescence for localization studies

• Immunoprecipitation to investigate protein-protein interactions

• Proximity labeling studies to identify interacting partners

• Monitoring SNX14 expression changes in disease models

• Validating genetic knockdown or knockout models

The HRP conjugation specifically provides enhanced sensitivity for detection methods like Western blotting and immunohistochemistry, eliminating the need for secondary antibody incubation steps .

How should researchers design Western blot experiments to detect SNX14 protein?

For optimal Western blot detection of SNX14 using HRP-conjugated antibodies:

• Sample preparation:

  • Extract proteins from tissues with high SNX14 expression (brain regions, lung, testis)

  • Use RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if investigating phosphorylated states

• Gel electrophoresis parameters:

  • Use 8-10% SDS-PAGE gels due to SNX14's size (~110 kDa)

  • Load 20-40 μg of total protein per lane

  • Include positive control samples from tissues with known SNX14 expression

• Transfer and detection:

  • Transfer to PVDF membranes (recommended over nitrocellulose)

  • Block with 5% non-fat milk or BSA in TBST

  • Dilute SNX14-HRP antibody 1:1000-1:5000 (optimize based on specific antibody)

  • Develop using ECL substrate with exposure times between 30 seconds to 5 minutes

• Controls to include:

  • Tissue-specific positive controls (hippocampal extracts)

  • Negative controls (heart tissue or SNX14 knockout samples)

What are effective immunoprecipitation protocols for studying SNX14 interactions?

An optimized immunoprecipitation protocol for SNX14:

• Lysis conditions:

  • Harvest cells in ice-cold lysis buffer containing 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), and protease/phosphatase inhibitors

  • For membrane-associated interactions, include 0.1% SDS and 0.5% sodium deoxycholate

  • Incubate on ice for 30 minutes with occasional vortexing

• Pre-clearing step:

  • Incubate lysates with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation (10,000 × g for 10 minutes)

• Immunoprecipitation:

  • Add SNX14 antibody (2-5 μg per 1 mg of protein lysate)

  • Incubate overnight at 4°C with gentle rotation

  • Add pre-washed Protein A/G beads and incubate for 2-4 hours

  • Wash 4-5 times with lysis buffer

  • Elute proteins with 2X SDS sample buffer

• Analysis:

  • Perform Western blotting for SNX14 and potential interacting proteins

  • For HRP-conjugated antibodies, special consideration is needed to avoid interference with the immunoprecipitation process

How can researchers quantify SNX14 expression levels across different experimental conditions?

For accurate quantification of SNX14 expression:

• RNA level quantification:

  • Perform RT-qPCR using SNX14-specific primers

  • Normalize expression to multiple housekeeping genes (GAPDH, β-actin, and 18S rRNA)

  • Calculate relative expression using the 2^-ΔΔCt method

• Protein level quantification:

  • Perform Western blotting using SNX14-HRP antibody

  • Include a concentration gradient of recombinant SNX14 for standard curve generation

  • Use digital imaging systems for densitometric analysis

  • Normalize to total protein (using stain-free technology) rather than single housekeeping proteins

• Considerations for accuracy:

  • Process all samples simultaneously under identical conditions

  • Repeat measurements across multiple biological replicates (n≥3)

  • Include positive and negative controls

  • Validate results using alternative detection methods when possible

How can SNX14-HRP antibodies be utilized in proximity labeling studies?

Proximity labeling with SNX14-HRP antibodies involves:

• Experimental setup:

  • Treat cells with biotin-phenol substrate for 30 minutes

  • Add H₂O₂ to initiate the HRP-catalyzed reaction (typically 1 mM for 1 minute)

  • Quench the reaction with antioxidants and sodium azide

  • Lyse cells and purify biotinylated proteins using streptavidin beads

• Optimization parameters:

  • Biotin-phenol concentration (0.1-1 mM)

  • H₂O₂ concentration (0.5-5 mM)

  • Reaction time (30 seconds to 5 minutes)

  • Cell density and growth conditions

• Analysis methods:

  • Western blotting with streptavidin-HRP to confirm biotinylation

  • Mass spectrometry to identify labeled proteins

  • Bioinformatic analysis to identify enriched pathways

• Validation approaches:

  • Confirm key interactions using co-immunoprecipitation

  • Perform reverse proximity labeling with identified partners

  • Use subcellular fractionation to verify compartment-specific interactions

This technique has revealed SNX14's role in saturated fatty acid metabolism and its interactions within the endoplasmic reticulum, providing new insights into its function.

What are the current methods for studying SNX14 mutations in relation to neurodevelopmental disorders?

To investigate SNX14 mutations associated with neurodevelopmental disorders:

• Genetic analysis approaches:

  • Whole exome sequencing (WES) to identify novel variants

  • Sanger sequencing for validation of identified mutations

  • ACMG guideline application for variant classification

  • Bioinformatic prediction tools (PolyPhen-2, SIFT, MutationTaster)

• Functional characterization methods:

  • Site-directed mutagenesis to introduce specific mutations (e.g., c.712A>T, c.2744A>T)

  • Transfection of mutant constructs into relevant cell lines

  • Western blotting with SNX14-HRP antibodies to assess protein expression

  • Immunofluorescence to determine subcellular localization changes

• Cellular phenotype assessments:

  • Analysis of lipid transport using fluorescent lipid analogs

  • Mitochondrial function assays (oxygen consumption, membrane potential)

  • ER stress response monitoring (BiP, CHOP expression)

  • Calcium homeostasis measurements

• Model systems:

  • Patient-derived fibroblasts or lymphoblasts

  • CRISPR/Cas9-modified cell lines

  • iPSC-derived neurons

  • Mouse models with equivalent mutations

How can researchers investigate the role of SNX14 in G-protein signaling pathways?

To study SNX14's function in G-protein signaling:

• Protein-protein interaction analysis:

  • Co-immunoprecipitation of SNX14 with Gαs proteins

  • GST pulldown assays using purified RGS domain of SNX14

  • Competitive binding assays between SNX14, Gαs, and 5-HT6R

  • FRET or BRET assays to measure real-time interactions

• Functional signaling assays:

  • cAMP accumulation assays following 5-HT stimulation

  • GTP single-turnover assays to assess GTPase activity

  • Calcium mobilization measurements

  • ERK phosphorylation detection as downstream readout

• Structural studies:

  • Identify key binding interfaces between SNX14 RGS domain and Gαs

  • Analyze effects of mutations on protein-protein interactions

  • Assess conformational changes using limited proteolysis

• Dynamic regulation analysis:

  • Investigate PKA-mediated phosphorylation of SNX14 at S382 and S388

  • Monitor translocation of SNX14 to plasma membrane using live-cell imaging

  • Assess effects of phosphorylation on Gαs binding versus 5-HT6R binding

What are common issues encountered when using SNX14-HRP antibodies and how can they be resolved?

When troubleshooting:

• Always include appropriate positive controls (brain tissue extracts)

• Perform antibody validation using SNX14 knockdown/knockout samples

• Test multiple antibody dilutions to determine optimal concentration

• Consider tissue/cell-specific expression patterns when interpreting results

How should researchers interpret contradictory data regarding SNX14 expression or function?

When facing contradictory SNX14 data:

• Systematic assessment approach:

  • Compare experimental conditions across studies (cell types, treatments, detection methods)

  • Evaluate antibody specificity and validation methods

  • Consider splice variant detection versus full-length protein

  • Assess post-translational modifications that might affect detection

• Validation strategies:

  • Use multiple detection methods (Western blot, qPCR, immunofluorescence)

  • Employ alternative antibodies targeting different epitopes

  • Include genetic manipulation (siRNA, CRISPR) as controls

  • Perform rescue experiments to confirm specificity

• Context-specific considerations:

  • Cell/tissue-specific expression patterns may explain differences

  • Developmental stage variations might account for contradictions

  • Subcellular localization can differ based on cellular conditions

  • Compensatory mechanisms may occur in different model systems

• Reporting recommendations:

  • Clearly document methodological details

  • Report all validation steps performed

  • Acknowledge limitations and potential alternative interpretations

  • Discuss findings in context of existing literature with potential explanations for discrepancies

What controls are essential when using SNX14-HRP antibodies in experimental research?

Essential controls for SNX14-HRP antibody experiments:

• Positive controls:

  • Tissue/cells known to express SNX14 (hippocampus, cerebellum, HT-22 cells)

  • Recombinant SNX14 protein (full-length or domain-specific)

  • Overexpression systems with tagged SNX14 constructs

• Negative controls:

  • Tissues with minimal SNX14 expression (heart, muscle)

  • SNX14 knockout/knockdown samples

  • Pre-absorption of antibody with immunizing peptide

  • Secondary antibody-only controls (for non-direct HRP methods)

• Specificity controls:

  • Detection of both endogenous and overexpressed SNX14

  • Comparison with alternative SNX14 antibodies

  • Western blot confirmation of immunofluorescence patterns

  • Size verification against predicted molecular weight

• Experimental validation:

  • Demonstrate expected changes following relevant treatments

  • Show consistency across multiple experimental replicates

  • Include loading/normalization controls for quantitative analyses

  • Verify subcellular localization patterns match known distribution

How are SNX14 antibodies contributing to our understanding of neurodevelopmental disorders?

SNX14 antibodies have been instrumental in elucidating the molecular mechanisms underlying SNX14-associated neurodevelopmental disorders:

• Genotype-phenotype correlations:

  • Western blot analysis using SNX14-HRP antibodies has revealed how different mutations affect protein expression levels

  • Immunohistochemistry has helped map SNX14 distribution in affected brain regions

  • Quantitative analysis has established connections between SNX14 levels and clinical severity

• Molecular pathways identification:

  • Proximity labeling studies have uncovered novel SNX14 interacting partners

  • Co-immunoprecipitation experiments have revealed disrupted protein interactions in disease states

  • Phosphorylation-specific antibodies have shown altered post-translational regulation

• Cellular pathology insights:

  • Immunofluorescence studies have demonstrated abnormal subcellular localization

  • Organelle-specific co-localization has revealed defects in lipid metabolism

  • Time-course analysis has shown developmental expression patterns relevant to disease onset

• Therapeutic target validation:

  • Antibody-based screening systems for potential drug candidates

  • Monitoring SNX14 levels/localization in response to experimental treatments

  • Assessment of restored protein function following genetic interventions

Recent studies have specifically linked SNX14 mutations to SCAR20 (spinocerebellar ataxia, autosomal recessive 20), with SNX14-HRP antibodies helping to demonstrate how novel variants (e.g., c.712A>T and c.2744A>T) affect protein expression and function.

What techniques are being developed to study SNX14's role in lipid metabolism?

Emerging techniques for investigating SNX14's function in lipid metabolism include:

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize SNX14-lipid interactions

  • Live-cell imaging with fluorescent lipid probes

  • Correlative light and electron microscopy for ultrastructural analysis

  • FRET-based sensors for lipid transfer activities

• Lipidomic analyses:

  • LC-MS/MS profiling of lipid species in SNX14-deficient models

  • Stable isotope labeling to track lipid trafficking pathways

  • Lipid droplet isolation and characterization

  • Targeted analysis of phosphoinositide species interacting with PX domain

• Genetic manipulation systems:

  • Domain-specific mutants to dissect lipid-binding functions

  • Inducible expression systems for temporal control

  • Cell-type specific conditional knockouts in animal models

  • CRISPR screens for SNX14-dependent lipid regulators

• Biochemical assays:

  • In vitro lipid binding assays with purified SNX14 domains

  • Membrane reconstitution systems

  • Lipid extraction efficiency measurements

  • ER stress response assays following lipid challenges

Current evidence suggests SNX14 deficiency leads to defective ER homeostasis and altered lipid saturation profiles, particularly following exposure to saturated fatty acids. This may explain the neuronal lipotoxicity and mitochondrial dysfunction observed in SNX14-related disorders.

How can researchers utilize SNX14 antibodies to investigate its interaction with serotonin receptors?

For studying SNX14-serotonin receptor interactions:

• Co-localization analysis:

  • Dual immunofluorescence labeling with SNX14 and 5-HT6R antibodies

  • Proximity ligation assays to detect direct interactions

  • FRET/BRET analysis for real-time interaction dynamics

  • Super-resolution microscopy to resolve subcellular interaction sites

• Functional interaction studies:

  • Monitor 5-HT6R surface expression using biotinylation assays

  • Track receptor internalization rates using antibody feeding assays

  • Measure receptor degradation with cycloheximide chase experiments

  • Assess downstream signaling pathways (cAMP production, ERK activation)

• Domain mapping approaches:

  • Generate domain deletion constructs to identify critical interaction regions

  • Study competition between SNX14, Gαs, and 5-HT6R using purified components

  • Investigate the impact of PKA-mediated phosphorylation on these interactions

  • Perform mutagenesis of key residues (S382, S388) to assess functional consequences

• Physiological relevance assessment:

  • Investigate SNX14-5-HT6R interactions in primary neurons

  • Correlate interaction dynamics with serotonin-dependent behaviors

  • Study the impact of disease-associated SNX14 mutations on receptor function

  • Develop small molecule modulators of the interaction