SNX14 Antibody, Biotin conjugated

What is SNX14 protein and why is it important in cellular research?

SNX14 is a sorting nexin family protein containing multiple functional domains, including a Phox (PX) domain and a regulator of G protein signaling (RGS) domain. It functions as a bifunctional negative regulator in serotonergic signaling pathways, particularly for the 5-hydroxytryptamine subtype 6 receptor (5-HT6R) . SNX14 is highly expressed in specific brain regions including the hippocampus, nucleus accumbens, and cerebellum, with additional expression in lung and testis tissues . Its importance in research stems from its dual regulatory role in receptor trafficking and G-protein signaling, as well as its association with distinctive autosomal-recessive cerebellar ataxia, intellectual disability, and coarsening facial features syndrome . Understanding SNX14 function is critical for research into neuronal excitability, synaptic transmission, and intracellular trafficking .

What is the purpose of using biotin-conjugated antibodies in SNX14 research?

Biotin-conjugated antibodies provide significant advantages in SNX14 detection systems due to the high-affinity interaction between biotin and avidin/streptavidin. This conjugation enables:

• Enhanced sensitivity in detection systems through signal amplification

• Versatile application across multiple detection platforms (microscopy, flow cytometry, ELISA)

• Compatibility with complex tissue samples where direct labeling might be problematic

• Ability to perform multi-step detection protocols with reduced background

Antibodies can be conjugated to biotin through various chemical methods, creating stable linkages that maintain antibody functionality while enabling interaction with avidin-coupled secondary reagents . For SNX14 research specifically, biotin-conjugated antibodies facilitate the detection of low-abundance SNX14 protein in neuronal tissues and allow for sensitive visualization of protein localization and trafficking dynamics .

What are the key structural domains of SNX14 and their functions?

SNX14 contains several distinct functional domains that contribute to its cellular roles:

• Regulator of G protein signaling (RGS) domain: Unlike related proteins, SNX14's RGS domain does not possess GAP (GTPase-activating protein) activity for Gαs. Instead, it specifically binds to and sequesters Gαs, thereby inhibiting 5-HT6R-mediated signaling pathways .

• Phox (PX) domain: Responsible for binding to membrane phosphoinositides, particularly phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a key component of late endosomes/lysosomes. Notably, SNX14 does not bind phosphatidylinositol 3-phosphate (PtdIns(3)P) .

• N-terminal hydrophobic region: Contributes to membrane association and protein-protein interactions .

• PXA domain: Present in the protein structure, though its specific function is still being investigated .

These domains work in concert to facilitate SNX14's role in endosomal trafficking, autophagosome clearance, and G-protein signaling regulation. The phosphoinositide-binding property of the PX domain is particularly crucial, as deletion mutants lacking this domain (ΔPX) fail to induce 5-HT6R internalization .

How do mutations in SNX14 relate to human disease?

Mutations in SNX14 cause a distinctive autosomal-recessive syndrome characterized by:

• Moderate to severe intellectual disability

• Cerebellar ataxia and early-onset cerebellar atrophy

• Sensorineural hearing loss

• Progressive coarsening of facial features

• Relative macrocephaly

• Notably, absence of seizures

Multiple types of mutations have been identified, including homozygous nonsense mutations, in-frame multiexon deletions, and splice site mutations . These mutations result in loss of normal biological function for SNX14, with affected individuals showing significantly reduced levels of SNX14 expression or production of truncated proteins . The specific mechanisms linking SNX14 dysfunction to the neurological and developmental phenotypes involve disruptions in autophagosome clearance and possibly fusion of lysosomes with autophagosomes .

What are the optimal methods for detecting SNX14 protein expression using biotin-conjugated antibodies?

For optimal detection of SNX14 protein using biotin-conjugated antibodies, researchers should consider a tiered approach based on the specific research question:

For Western Blotting:

• Extraction protocol: Use a lysis buffer containing adequate detergent concentration to solubilize membrane-associated SNX14

• Expected molecular weight: ~110 kDa for wild-type protein

• Detection system: Streptavidin-HRP or streptavidin-conjugated fluorophores

• Controls: Include positive controls from tissues with known high expression (hippocampus, cerebellum) and negative controls from tissues with low expression (heart, muscle)

For Immunohistochemistry/Immunofluorescence:

• Fixation: 4% paraformaldehyde is recommended for preserving SNX14 epitopes

• Antigen retrieval: May be necessary for formalin-fixed tissues

• Blocking: Include avidin/biotin blocking steps to reduce endogenous biotin interference

• Signal amplification: Consider tyramide signal amplification for low-abundance detection

• Colocalization markers: Include markers for endosomal compartments (for trafficking studies) or G-protein signaling components (for signaling studies)

For Flow Cytometry:

• Permeabilization: Required for intracellular SNX14 detection

• Titration: Determine optimal antibody concentration to minimize background

• Multiparameter analysis: Consider combining with markers for neuronal subtypes or activation states

The choice of detection method should be informed by the specific subcellular localization being investigated, as SNX14 shows dynamic trafficking between cytoplasmic punctate structures (endosome-like) and plasma membrane upon 5-HT stimulation .

How does SNX14 regulate 5-HT6 receptor trafficking and signaling?

SNX14 functions as a dual negative regulator of 5-HT6 receptor through two distinct mechanisms:

Mechanism 1: Receptor Trafficking Regulation

• SNX14 accelerates the internalization and degradation of 5-HT6R through its RGS-PX domain

• Expression of the RGS-PX domain decreases both total and surface 5-HT6R levels

• This effect is specific to 5-HT6R and not observed with other receptors like β2AR

• The phosphoinositide-binding property of the PX domain is essential for this function, as ΔPX mutants fail to induce 5-HT6R internalization

• Upon 5-HT treatment, endogenous SNX14 is recruited to the plasma membrane

• TIRF microscopy reveals that SNX14 and 5-HT6R are co-internalized following 5-HT stimulation

Mechanism 2: G-protein Signaling Inhibition

• Despite containing an RGS domain, SNX14 does not possess GAP activity for Gαs

• Instead, SNX14 specifically binds to activated Gαs and sequesters it

• This sequestration prevents Gαs from activating adenylyl cyclase, thereby inhibiting cAMP production

• The RGS domain of SNX14 competitively binds to the intracellular loop 3 (iL3) region of 5-HT6R, interfering with Gαs binding

• Phosphorylation of the RGS domain by protein kinase A (PKA) regulates the binding affinity of SNX14 for Gαs

These two mechanisms work in concert to comprehensively down-regulate 5-HT6R signaling: SNX14 both reduces receptor availability at the cell surface and attenuates downstream signal transduction from remaining receptors.

What are the recommended experimental controls when using biotin-conjugated SNX14 antibodies?

To ensure reliable and interpretable results when using biotin-conjugated SNX14 antibodies, the following controls should be implemented:

Antibody Specificity Controls:

• Genetic validation: Compare staining/detection between wild-type samples and SNX14 knockout/knockdown samples

• Peptide competition: Pre-incubate antibody with immunizing peptide (601-893AA of SNX14) to demonstrate specific binding

• Isotype control: Include a biotin-conjugated isotype-matched irrelevant antibody

• Secondary-only control: Omit primary antibody to assess background from streptavidin reagents

Sample Preparation Controls:

• Positive tissue controls: Include tissues known to express SNX14 (hippocampus, cerebellum, cortex)

• Negative tissue controls: Include tissues with minimal SNX14 expression (heart, muscle)

• Stimulation controls: Compare 5-HT treated versus untreated samples when studying dynamic trafficking

Technical Controls:

• Endogenous biotin blocking: Apply avidin/biotin blocking reagents, particularly critical in biotin-rich tissues

• Titration series: Determine optimal antibody concentration by testing a range of dilutions

• Cross-reactivity assessment: Validate specificity against related sorting nexins (particularly SNX13, which shares structural similarities but has different tissue distribution)

Validation Controls:

• Orthogonal detection: Confirm findings using alternative detection methods (e.g., mass spectrometry)

• Subcellular fractionation: Verify localization findings by comparing detection across cellular compartments

• Multiple antibodies: When possible, confirm results with antibodies targeting different epitopes of SNX14

These comprehensive controls address the potential pitfalls in biotin-conjugated antibody applications, including endogenous biotin interference, non-specific binding, and inappropriate detection thresholds.

What methodologies can detect SNX14 translocation during receptor activation?

To effectively monitor the dynamic translocation of SNX14 during receptor activation (particularly upon 5-HT6R stimulation), several complementary methodologies can be employed:

Total Internal Reflection Fluorescence (TIRF) Microscopy

• Allows selective visualization of plasma membrane-proximal events

• Can track the recruitment of SNX14 to the membrane following 5-HT treatment

• Enables co-localization studies with Gαs and 5-HT6R

• Capable of detecting the subsequent re-internalization of SNX14

Surface Biotinylation and Membrane Protein Extraction

• Employs EZ-Link® Sulfo-NHS-SS-Biotin (0.25 mg/ml) to label surface proteins

• Requires 30 min incubation at 4°C followed by quenching and NeutraAvidin pulldown

• Can be combined with receptor activation protocols (e.g., 10 μM 5-HT treatment)

• For internalization studies, surface biotin can be cleaved with glutathione solution (50 mM glutathione, 75 mM NaCl, 75 mM NaOH in FBS)

Subcellular Fractionation with Immunoblotting

• Separates plasma membrane, cytosolic, and endosomal fractions

• Allows quantitative assessment of SNX14 redistribution following stimulation

• Should include markers for each compartment (Na+/K+ ATPase for plasma membrane, EEA1 for early endosomes)

Live-Cell Confocal Microscopy with Fluorescently Tagged Proteins

• Real-time visualization of SNX14 trafficking

• Can employ photoactivatable or photoconvertible tags for pulse-chase experiments

• Enables assessment of trafficking kinetics and protein-protein interactions

Proximity Ligation Assay (PLA)

• Detects protein-protein interactions between SNX14 and binding partners (Gαs, 5-HT6R)

• Provides spatial information about interaction locations

• Can track changes in interaction patterns following receptor activation

An integrated approach combining these methodologies provides comprehensive insights into the spatiotemporal dynamics of SNX14 translocation, its association with receptor complexes, and its role in receptor internalization.

How can researchers quantitatively assess SNX14-mediated receptor internalization?

Quantitative assessment of SNX14-mediated receptor internalization (particularly for 5-HT6R) requires a multi-faceted approach combining biochemical and imaging methodologies:

Biochemical Quantification:

• Surface Biotinylation Assay:

  • Label cell surface proteins with cleavable biotin (EZ-Link® Sulfo-NHS-SS-Biotin)

  • Stimulate cells with ligand (e.g., 10 μM 5-HT)

  • Remove remaining surface biotin with glutathione solution

  • Isolate internalized biotinylated proteins with NeutraAvidin

  • Quantify receptor levels by immunoblotting with receptor-specific antibodies

  • This approach specifically measures internalized receptor pools

• Flow Cytometry:

  • Label surface receptors with fluorescent antibodies before and after stimulation

  • Calculate internalization as percent decrease in surface fluorescence

  • Can be combined with SNX14 overexpression or knockdown to assess functional impact

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Develop sandwich ELISA with capture antibodies against extracellular receptor domains

  • Compare surface receptor levels across timepoints after stimulation

  • Use biotin-conjugated detection antibodies for enhanced sensitivity

Imaging-Based Quantification:

• Automated High-Content Imaging:

  • Immunolabel surface receptors before and after stimulation

  • Apply automated image analysis to quantify receptor distribution

  • Calculate internalization index (ratio of internal to surface fluorescence)

  • Compare kinetics between wild-type cells and those with SNX14 manipulation

• Pulse-Chase Fluorescence Microscopy:

  • Label surface receptors with pH-sensitive fluorescent conjugates

  • Track fluorescence changes as receptors move to acidic endosomal compartments

  • Calculate internalization rates from fluorescence intensity changes

Data Analysis Considerations:

• Kinetic Parameters:

  • Half-time of internalization (t1/2)

  • Maximum internalization (Bmax)

  • Initial rate of internalization

• Comparative Analysis:

  • Control vs. SNX14 overexpression

  • Control vs. SNX14 knockdown/knockout

  • Wild-type SNX14 vs. domain mutants (particularly ΔPX)

• Statistical Validation:

  • Determine significance through appropriate statistical tests

  • Account for cell-to-cell variability through sufficient replicates

  • Consider dose-response relationships to ligand concentration

This comprehensive approach enables precise quantification of SNX14's impact on receptor trafficking dynamics, providing insights into both the rate and extent of internalization processes.

What is the optimal protocol for conjugating SNX14 antibodies to biotin?

The optimal protocol for conjugating SNX14 antibodies to biotin balances efficiency of labeling with preservation of antibody function:

Materials Required:

• Purified SNX14 antibody (polyclonal or monoclonal)

• NHS-biotin or Sulfo-NHS-LC-biotin

• Dialysis cassettes or desalting columns

• PBS buffer (pH 7.4)

• 0.1M sodium bicarbonate buffer (pH 8.4)

• Glycerol for storage

Procedure:

• Antibody Preparation:

  • Ensure antibody concentration is between 1-10 mg/ml in PBS

  • If necessary, concentrate using centrifugal filters

  • Remove preservatives like sodium azide or glycerol by dialysis against PBS

• Biotin Conjugation:

  • Dissolve NHS-biotin in DMSO at 10 mg/ml

  • Add biotin reagent to antibody solution at 10-20 molar excess

  • Incubate for 2 hours at room temperature or overnight at 4°C

  • Reaction occurs via primary amines (lysine residues) on the antibody

• Purification:

  • Remove unreacted biotin by dialysis against PBS (3 changes)

  • Alternatively, use a desalting column equilibrated with PBS

  • For highest purity, consider gel filtration chromatography

• Quality Control:

  • Determine biotin incorporation ratio using HABA assay

  • Optimal labeling: 4-8 biotin molecules per antibody

  • Excessive biotinylation can compromise antigen binding

• Storage:

  • Add preservative (e.g., 0.03% Proclin 300)

  • Add stabilizer (50% glycerol)

  • Store at -20°C or -80°C to avoid repeated freeze-thaw cycles

Critical Considerations:

• pH control is essential: biotinylation efficiency peaks at pH 8.0-8.5

• Temperature affects reaction rate but can impact antibody stability

• The spacer arm length in LC-biotin derivatives improves accessibility to avidin/streptavidin

• Optimize biotinylation ratio for each application (ELISA vs. immunohistochemistry)

This protocol ensures production of consistently labeled SNX14 antibodies suitable for sensitive detection systems while maintaining specificity and binding affinity.

How can researchers validate mutations or expression changes in SNX14?

Comprehensive validation of SNX14 mutations or expression changes requires a multi-modal approach combining molecular, protein-level, and functional analyses:

DNA-Level Validation:

• Sanger Sequencing:

  • Direct sequencing of SNX14 coding regions

  • Particularly useful for confirming point mutations, small insertions/deletions

  • Can detect splice site mutations as seen in some SNX14-related disorders

• Next-Generation Sequencing:

  • Whole exome/genome sequencing for comprehensive mutation detection

  • RNA-seq to identify altered splicing patterns

  • Can identify complex structural variants like multi-exon deletions

• PCR-Based Methods:

  • Real-time PCR to validate copy number variations

  • Long-range PCR to characterize large deletions or insertions

  • RT-PCR to detect aberrant splicing events

RNA-Level Validation:

• Quantitative RT-PCR:

  • Compare mutant and wild-type transcript levels

  • Useful for detecting nonsense-mediated decay

  • Has demonstrated significantly reduced levels in some SNX14 mutations

• Northern Blotting:

  • Visualize transcript size changes

  • Detect multiple splice variants

  • Less sensitive but provides direct size information

Protein-Level Validation:

• Immunoblotting:

  • Detect presence/absence of SNX14 protein

  • Identify truncated proteins or altered molecular weight

  • Wild-type SNX14 appears as ~110 kDa band

  • Mutations may result in no detectable protein or altered size (~107 kDa for exon 19 skipping)

• Mass Spectrometry:

  • Precise characterization of protein modifications

  • Identification of altered peptide fragments

  • Quantitative comparison of protein levels

Functional Validation:

• cAMP Production Assays:

  • Measure impact on 5-HT-induced cAMP elevation

  • Compare with forskolin-induced responses

  • Assess negative regulatory function of SNX14

• Receptor Trafficking Assays:

  • Surface biotinylation to measure receptor internalization

  • Fluorescence imaging to track receptor localization

  • Compare wild-type with mutant SNX14 effects

• Protein Interaction Studies:

  • Co-immunoprecipitation to assess binding to Gαs and 5-HT6R

  • Pulldown assays with GST-tagged domains

  • Interaction with activated vs. inactive G proteins

This comprehensive validation approach ensures accurate characterization of SNX14 mutations and their functional consequences, critical for both research applications and clinical correlations.

What are the key challenges and future directions in SNX14 antibody research?

Current challenges and emerging opportunities in SNX14 antibody research span technical, biological, and translational domains:

Technical Challenges:

• Specificity validation: Developing antibodies that can distinguish between closely related sorting nexin family members

• Epitope accessibility: Optimizing detection of conformational changes during SNX14's dynamic trafficking

• Quantitative reproducibility: Standardizing detection methods across different experimental systems

• Tissue penetration: Improving antibody performance in complex neural tissues where SNX14 functions

Biological Research Frontiers:

• Cell-type specific functions: Characterizing SNX14 roles across different neuronal populations

• Post-translational modifications: Mapping phosphorylation and other modifications that regulate SNX14 activity

• Protein complex dynamics: Identifying the complete interactome of SNX14 beyond Gαs and 5-HT6R

• Developmental regulation: Understanding SNX14 expression patterns during brain development and maturation

Emerging Applications:

• Patient-derived models: Using biotin-conjugated SNX14 antibodies to characterize cellular phenotypes in samples from individuals with SNX14 mutations

• High-throughput screening: Developing assays to identify compounds that modulate SNX14 function

• In vivo imaging: Adapting antibody-based detection for real-time visualization of SNX14 dynamics

• Single-cell analysis: Integrating SNX14 detection into multi-parameter single-cell profiling technologies

Translational Opportunities:

• Biomarker development: Exploring SNX14 as a potential biomarker for specific neurological conditions

• Therapeutic targeting: Leveraging understanding of SNX14 function to develop interventions for associated disorders

• Diagnostic applications: Refining SNX14 mutation detection and functional characterization for clinical diagnostics

• Model systems: Developing and validating animal and cellular models that recapitulate SNX14-associated disorders