STOML3 Antibody

What is STOML3 and where is it expressed?

STOML3 (Stomatin-like protein-3) is an integral membrane protein belonging to the stomatin-domain family of proteins. It is primarily expressed in mechanosensory neurons of dorsal root ganglia (DRG) and olfactory sensory neurons (OSNs) . In OSNs, STOML3 is enriched in the knob and proximal part of the cilia . The protein contains a conserved stomatin-domain core and is inserted into the membrane via a short hydrophobic hairpin located at the N-terminus .

What are the recommended applications for STOML3 antibodies?

STOML3 antibodies are validated for multiple research applications:

• Western Blotting (WB): Generally recommended at dilutions of 1:500-1:2000

• Immunocytochemistry/Immunofluorescence (ICC/IF): Validated in multiple cell lines

• Immunohistochemistry (IHC): Used in tissue sections with appropriate antigen retrieval methods

• ELISA: Validated with specific antibody formulations

What species reactivity is observed with STOML3 antibodies?

Most commercial STOML3 antibodies show reactivity with human, mouse, and rat samples . Some antibodies have broader reactivity profiles including:

• Cow, Guinea Pig, Rabbit, Horse, and Monkey

• High percent identity (92-100%) across many species including Dog, Pig, and other mammals

How can I verify the specificity of STOML3 antibodies?

Specificity of STOML3 antibodies can be verified through:

• Testing on HEK-293 cells transiently transfected with plasmids containing STOML3 cDNA fused with EGFP or mCherry

• Using knockout models (Stoml3 KO mice) as negative controls

• Comparing detection patterns with the predicted molecular weight (approximately 32 kDa, though observed at approximately 30 kDa)

• Validating across multiple applications (WB, ICC/IF, IHC) to ensure consistent detection patterns

How does STOML3 modulate mechanosensitive channels, and can antibodies help investigate this function?

STOML3 modulates mechanosensitive channels through association with cholesterol in the membrane. This association creates a stiffened membrane platform that facilitates force transfer to channels like Piezo1 and Piezo2 . To investigate this function:

• Use STOML3 antibodies in co-immunoprecipitation studies to examine protein-protein interactions between STOML3 and mechanosensitive channels

• Employ immunohistochemistry with STOML3 antibodies alongside cholesterol-binding probes to visualize co-localization in lipid rafts

• Combine STOML3 antibody staining with atomic force microscopy (AFS) to correlate STOML3 expression with membrane mechanical properties

The proline-40 residue is critical for STOML3's cholesterol binding and subsequent modulation of mechanosensitive channels. The STOML3-P40S mutant fails to associate with cholesterol-rich lipid rafts and cannot potentiate Piezo1 or Piezo2 currents .

What are the methodological considerations when using STOML3 antibodies for detecting subcellular localization?

For optimal detection of STOML3's subcellular localization in neurons and sensory cells:

• Fixation protocol: PFA fixation with Triton X-100 permeabilization is recommended

• Antigen retrieval: For tissues with cross-linking fixatives, sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) heated at 100°C for 5 minutes improves antibody access

• Signal amplification: For low abundance detection, employ tyramide signal amplification methods such as the Tyramide SuperBoost kit

• Co-localization studies: STOML3 shows a punctate intracellular pattern in OSNs that co-localizes with the endosomal marker Rab11, indicating vesicular localization

• Validation controls: Include both Stoml3 KO tissues and Triple KO (where Stom, Stoml1, and Stoml3 are knocked out) for comprehensive specificity validation

How do STOML3 antibodies perform in comparative analysis across the stomatin family?

When conducting comparative analysis across the stomatin family:

• Specificity is crucial due to sequence homology between family members (STOM, STOML1, STOML2, STOML3, and podocin)

• Commercial antibodies for STOML3 should be validated against other family members by using cells transfected with each specific family member

• The conserved proline residue (P40 in STOML3) is present across stomatin family proteins and is critical for cholesterol binding

• Consider using panel approaches that include antibodies against multiple stomatin family members to examine differential expression or compensatory mechanisms in knockout models

What strategies can resolve contradictory STOML3 antibody data in expression studies?

When facing contradictory results with STOML3 antibodies:

• Epitope considerations: Use antibodies targeting different epitopes of STOML3 (e.g., N-terminal region aa 1-50 vs. middle region aa 143-192 vs. C-terminal region aa 205-291)

• Sample preparation variations: Different extraction methods may affect membrane protein solubilization and epitope accessibility

• Protein modification effects: Consider post-translational modifications that might mask epitopes

• Expression level sensitivity: For low-expressing tissues, compare amplification methods with more sensitive detection systems like immunoaffinity purified antibodies

• Signal verification: Validate signals using genetic models (Stoml3 KO) to distinguish specific from non-specific signals, as some antibodies may show staining in KO models that represents cross-reactivity

What is the optimal experimental design for investigating STOML3-cholesterol interactions using antibodies?

To investigate STOML3-cholesterol interactions:

• Lipid raft isolation: Use sucrose density gradient ultracentrifugation followed by Western blotting with STOML3 antibodies to detect raft association

• Cholesterol depletion studies: Compare STOML3 localization before and after methyl-β-cyclodextrin (MβCD) treatment using immunofluorescence

• Mutagenesis analysis: Generate P40S mutant of STOML3 (which disrupts cholesterol binding) and compare antibody staining patterns between wild-type and mutant STOML3

• Force measurements: Combine immunofluorescence with atomic force spectroscopy (AFS) to correlate STOML3 expression with membrane stiffness properties

• Functional readouts: Use electrophysiological recordings in cells expressing STOML3 to correlate antibody-detected expression levels with mechanosensitive current amplitudes

How can STOML3 antibodies be used to investigate its role in sensory neurons?

For investigating STOML3's role in sensory neurons:

• Neuronal type identification: Use double immunostaining with STOML3 antibodies and neuronal markers to identify specific sensory neuron populations (e.g., mechanoreceptors or olfactory neurons)

• Subcellular localization: Employ high-resolution imaging with STOML3 antibodies to detect expression in specialized structures like dendritic knobs and cilia of OSNs

• Functional correlation: Combine loose patch recordings of spontaneous and stimulus-induced firing with post-recording immunostaining to correlate STOML3 expression with neuronal activity

• In vivo relevance: Use behavioral assays (such as mechanical sensitivity tests) in conjunction with tissue analysis using STOML3 antibodies to link molecular findings to physiological outcomes

What controls should be included when using STOML3 antibodies in knockout or knockdown studies?

Essential controls for STOML3 antibody studies in knockout or knockdown contexts:

• Genetic verification: Confirm genetic modification at DNA and RNA levels before proceeding to protein detection

• Positive controls: Include wild-type samples alongside knockout/knockdown samples

• Specificity controls: Include single knockout (Stoml3 KO) and multiple knockout (Triple KO with Stom, Stoml1, and Stoml3) to distinguish specific from non-specific signals

• Rescue experiments: Transfect knockout cells with wild-type STOML3 to restore expression and confirm antibody specificity

• Cross-reactivity assessment: Test antibodies against related proteins (other stomatin family members) to ensure specificity

• Alternative antibodies: Use multiple antibodies targeting different epitopes of STOML3 to confirm findings

How can I optimize Western blotting conditions for STOML3 antibodies?

For optimal Western blotting with STOML3 antibodies:

• Sample preparation: Use appropriate lysis buffers containing detergents suitable for membrane proteins (e.g., RIPA or NP-40)

• Loading controls: Include appropriate loading controls for membrane proteins (not just cytosolic proteins like GAPDH)

• Gel percentage: Use 10-12% SDS-PAGE gels for optimal resolution around the expected molecular weight (32 kDa)

• Transfer conditions: Optimize transfer conditions for membrane proteins (lower methanol concentration may help)

• Blocking agent: Test different blocking agents (BSA vs. milk) as some antibodies perform better with specific blockers

• Antibody concentration: Start with the recommended dilution (1:500-1:2000) and optimize based on signal-to-noise ratio

• Expected band size: Look for bands at approximately 30-32 kDa, the predicted molecular weight for STOML3

What strategies address non-specific binding issues with STOML3 antibodies?

To address non-specific binding:

• Increase washing stringency: Use higher concentrations of Tween-20 (0.1-0.3%) in wash buffers

• Optimize blocking: Extend blocking time or test alternative blocking agents

• Pre-adsorption: Pre-incubate antibody with the immunizing peptide to identify specific bands

• Increase antibody specificity: Use immunoaffinity purified antibodies rather than crude sera

• Knockout validation: Compare staining patterns between wild-type and Stoml3 KO samples to identify non-specific signals

• Dilution optimization: Test a range of antibody dilutions to find the optimal concentration that maximizes specific signal while minimizing background

What are the critical parameters for immunofluorescence studies with STOML3 antibodies?

Critical parameters for immunofluorescence with STOML3 antibodies:

• Fixation method: PFA fixation (typically 4%) with Triton X-100 permeabilization works well for most applications

• Antigen retrieval: For tissue sections, sodium citrate buffer heating may be necessary to expose epitopes

• Signal amplification: Consider tyramide signal amplification for detecting low-abundance STOML3

• Blocking duration: Extended blocking (1-2 hours) may reduce background

• Primary antibody incubation: Overnight incubation at 4°C often yields optimal results

• Expected patterns: Look for punctate staining in cell bodies and enrichment in specialized structures such as dendritic knobs and proximal cilia in OSNs

• Counterstaining: Include membrane markers to confirm proper localization of STOML3 to membrane compartments

How should I interpret conflicting data between different STOML3 antibody applications?

When interpreting conflicting data between applications:

• Application-specific optimization: Each application (WB, IF, IHC) may require specific optimization for the same antibody

• Epitope accessibility: Different sample preparations may expose or mask epitopes differently

• Protein conformation: Native vs. denatured protein may affect antibody recognition

• Cross-validation: Use multiple antibodies targeting different STOML3 epitopes to confirm findings

• Functional validation: Correlate antibody detection with functional assays (e.g., mechanosensitivity measurements)

• Genetic models: Use knockout models as definitive controls to resolve conflicting results

• Methodological variations: Consider that different detection methods (fluorescence vs. chromogenic) may have different sensitivity thresholds

How can STOML3 antibodies facilitate research into membrane mechanics and ion channel function?

STOML3 antibodies can advance research into membrane mechanics by:

• Co-localization studies: Visualize STOML3 in relation to Piezo1/2 and other mechanosensitive channels

• Membrane microdomain analysis: Combine with cholesterol staining to identify specialized membrane microdomains

• Structure-function analysis: Compare wild-type STOML3 with P40S mutant localization to understand cholesterol binding domains

• Biophysical correlations: Use immunolabeling intensity to correlate STOML3 expression levels with membrane stiffness measurements from atomic force microscopy

• Molecular mechanism studies: Investigate how STOML3-mediated changes in membrane mechanics modulate ion channel sensitivity using antibodies to track protein expression and localization

What are the considerations for using STOML3 antibodies in studying sensory pathologies?

For studying sensory pathologies:

• Expression analysis: Compare STOML3 expression levels between normal and pathological tissues using calibrated immunostaining approaches

• Localization changes: Examine potential redistribution of STOML3 in disease states using subcellular fractionation followed by Western blotting

• Animal models: Use STOML3 antibodies to characterize expression in animal models of sensory disorders, particularly mechanical allodynia

• Therapeutic target validation: Monitor STOML3 expression after experimental therapies targeting the STOML3-cholesterol association

• Clinical correlation: Correlate STOML3 expression patterns with sensory function in patient samples using standardized immunohistochemistry protocols

How can researchers quantitatively assess STOML3 expression using antibodies?

For quantitative assessment of STOML3 expression:

• Western blot densitometry: Normalize STOML3 band intensity to appropriate loading controls

• Quantitative immunofluorescence: Use standardized image acquisition settings and quantify fluorescence intensity relative to reference standards

• Flow cytometry: For cell populations, quantify STOML3 antibody binding using flow cytometry with appropriate permeabilization protocols

• ELISA development: Develop sandwich ELISA using capture and detection antibodies against different STOML3 epitopes

• Mass spectrometry validation: Combine immunoprecipitation using STOML3 antibodies with mass spectrometry for absolute quantification

• Single-cell analysis: Combine STOML3 immunostaining with single-cell electrophysiology to correlate expression with functional properties