STOML1 Antibody

What is STOML1 and why is it important for research?

STOML1 (Stomatin-like protein 1) is a membrane protein with a characteristic bipartite structure containing a stomatin domain and a sterol carrier protein-2 (SCP-2) domain. This unique structure suggests a role in sterol/lipid transfer and transport. STOML1 is predominantly expressed in the brain, with lower levels detected in other tissues . The protein is significant for research because:

• It localizes to late endosomal compartments and interacts with stomatin

• It may play a role in cholesterol transfer to late endosomes

• It can modulate membrane acid-sensing ion channels, specifically inhibiting proton-gated current of ASIC1 isoform 1 and increasing inactivation speed of ASIC3

• It may be involved in regulation of proton sensing in dorsal root ganglions

• It potentially protects FBXW7 isoform 3 from degradation

Understanding STOML1 function provides insights into cellular lipid distribution, membrane protein trafficking, and ion channel regulation.

What are the key features of STOML1 antibodies available for research?

Research-grade STOML1 antibodies typically share these characteristics:

When selecting an antibody, researchers should consider the specific epitope targeted and validated applications relevant to their experimental design.

How is STOML1 expressed in different tissues and cell types?

STOML1 expression follows a specific pattern across tissues:

• Highest expression occurs in brain tissue

• It is ubiquitously expressed at low levels across other tissues

• Within the olfactory epithelium, STOML1 shows low expression levels primarily in the cell body of mature olfactory sensory neurons (OSNs) and is absent from the ciliary layer

• STOML1 is detected in a subpopulation of sensory neurons in the dorsal root ganglia

• In expression studies, the protein localizes specifically to late endosomal compartments and is absent from the plasma membrane, unlike other stomatin family members

This expression pattern suggests tissue-specific functions, particularly in neuronal systems where STOML1 may participate in specialized membrane trafficking and signaling.

How should I design experiments to study STOML1 interactions with stomatin and other binding partners?

To effectively study STOML1 interactions:

• Co-immunoprecipitation approach: Design co-IP experiments using anti-STOML1 antibodies to pull down protein complexes, followed by detection of potential binding partners like stomatin. Research has shown that STOML1 and stomatin co-immunoprecipitate, indicating direct interaction .

• Fluorescent co-localization studies: Use dual immunofluorescence with anti-STOML1 and anti-stomatin antibodies. Studies have demonstrated co-localization in late endosomal compartments .

• Membrane fractionation: Isolate detergent-resistant membranes (DRMs) to analyze association of STOML1 with lipid rafts, as both STOML1 and stomatin associate with DRMs .

• Mutational analysis: Create deletion mutants of STOML1 and assess their impact on protein-protein interactions. For example, the GYxxΦ sorting signal at the N-terminus is critical for late endosomal targeting .

• Live-cell imaging: Express fluorescently-tagged STOML1 to monitor dynamic interactions with other proteins. This has been successfully used to show that overexpression of STOML1 leads to redistribution of stomatin from the plasma membrane to late endosomes .

Control experiments should include knockout/knockdown models, such as the Triple KO mouse line (where Stom, Stoml1, and Stoml3 are knocked out) and the Stoml3 KO mouse line, which have been used to validate antibody specificity in immunostaining analyses .

What are the most effective methods for validating STOML1 antibody specificity?

Comprehensive validation of STOML1 antibodies should include multiple approaches:

• Western blot with positive controls: Use tissues known to express STOML1, such as brain samples, A375 cells, or Molt-4 cells . A specific antibody should detect a band at approximately 45 kDa.

• Knockout validation: Test antibodies on samples from STOML1 knockout models. Studies have used Triple KO mouse lines (lacking Stom, Stoml1, and Stoml3) to confirm antibody specificity - the absence of staining in these models validates specificity .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide/protein. For example, STOML1 antibodies pre-incubated with Stomatin-like Protein 1 blocking peptide show eliminated staining in western blots of rat dorsal root ganglia and mouse brain lysates .

• Heterologous expression systems: Test antibodies on HEK-293 cells transiently transfected with plasmids containing STOML1 cDNA sequences. Research has shown these transfection systems are useful for confirming selectivity within the stomatin-domain protein family .

• Cross-reactivity assessment: Test the antibody against related proteins (STOM, STOML2, STOML3) to ensure specificity within the stomatin family.

A comprehensive validation should include at least three of these approaches to ensure antibody specificity before proceeding with experimental applications.

What are the critical considerations for designing immunohistochemistry experiments with STOML1 antibodies?

Successful immunohistochemistry with STOML1 antibodies requires attention to several key factors:

• Antigen retrieval optimization: For paraffin-embedded tissues, test both TE buffer pH 9.0 and citrate buffer pH 6.0. Research indicates that antigen retrieval conditions significantly impact STOML1 detection .

• Amplification systems: Consider using tyramide signal amplification methods, particularly for low-expression tissues. This approach has been successfully employed to reveal anti-STOML1 staining in the olfactory system .

• Dilution optimization: Start with a broad range (1:20-1:500) and optimize for your specific tissue. Most protocols recommend 1:50-1:500 for IHC applications .

• Proper controls:

  • Positive controls: Human brain tissue and gliomas tissue show reliable STOML1 expression

  • Negative controls: Use tissues from knockout models or tissues known to lack STOML1 expression

  • Absorption controls: Pre-incubate antibody with immunizing peptide

• Co-staining strategies: For neuronal tissues, consider co-staining with OMP (olfactory marker protein) to identify mature olfactory sensory neurons, as STOML1 has been shown to localize mainly in the cell body of OMP-positive neurons .

• Subcellular localization awareness: Remember that STOML1 shows a punctate intracellular pattern rather than membrane staining. This differs from stomatin, which also shows plasma membrane localization .

These considerations will help ensure specific and reproducible STOML1 detection in tissue sections.

How should I interpret conflicting results between different STOML1 antibodies?

When facing discrepancies between different STOML1 antibodies:

• Compare epitope targets: Antibodies targeting different regions of STOML1 may yield varying results. For example, antibodies targeting the N-terminal region (AA 1-30) versus the central region (AA 79-290) may have different access to epitopes depending on protein conformation or interactions .

• Evaluate application suitability: Some antibodies perform better in specific applications. For instance, an antibody might work well for western blotting but poorly for immunohistochemistry due to differences in protein denaturation states .

• Consider fixation and sample preparation effects: STOML1's membrane association and subcellular localization can be affected by different fixation methods. The protein's localization to late endosomes may be preserved differently depending on sample preparation .

• Assess antibody cross-reactivity: Despite validation, some antibodies may cross-react with other stomatin family members. The stomatin domain is conserved across family members (STOM, STOML1, STOML2, STOML3), potentially causing non-specific binding .

• Validate with orthogonal methods: If antibodies yield conflicting results, confirm findings using non-antibody methods such as mRNA expression analysis or tagged protein expression.

The most reliable interpretation comes from triangulating results using multiple antibodies targeting different epitopes and complementary methods like genetic models.

What are common pitfalls in analyzing STOML1 expression and localization data?

Researchers should be aware of these common pitfalls:

Awareness of these pitfalls can help researchers design more robust experiments and correctly interpret their results.

How do I troubleshoot non-specific binding or weak signal when using STOML1 antibodies?

When encountering problems with STOML1 antibody performance:

• For non-specific binding:

  • Increase blocking time and concentration (5% BSA with 1% normal serum from the secondary antibody species)

  • Optimize antibody dilution - try higher dilutions than recommended (e.g., 1:1000 instead of 1:500)

  • Add 0.05% Tween-20 to washing and antibody dilution buffers

  • For western blots, include multiple washes with higher salt concentration

  • Consider using more specific monoclonal antibodies if available

• For weak signal:

  • Try signal amplification methods like tyramide signal amplification, successfully used to reveal anti-STOML1 staining in the olfactory system

  • Optimize antigen retrieval methods - test both TE buffer pH 9.0 and citrate buffer pH 6.0

  • Increase antibody concentration or incubation time

  • For western blots, load more protein (50-100 μg total protein)

  • Consider using different antibodies targeting other epitopes of STOML1

  • For tissues with low expression, enrich the sample (e.g., subcellular fractionation focusing on membrane components)

• For both issues:

  • Verify sample quality and protein integrity

  • Check antibody storage conditions - aliquot and store at -20°C to avoid freeze/thaw cycles

  • Test fresh antibody lots, as antibody performance can deteriorate over time

  • Validate the antibody with known positive controls such as brain tissue, A375 cells, or Molt-4 cells

Systematic troubleshooting following this approach can help resolve most common issues with STOML1 antibodies.

What are the optimal protocols for using STOML1 antibodies in western blotting?

For optimal western blot results with STOML1 antibodies:

Sample Preparation:

• Extract proteins from tissues or cells using RIPA buffer supplemented with protease inhibitors

• For membrane-associated proteins like STOML1, consider membrane fractionation to enrich the target

• Load 30-50 μg of total protein per lane

SDS-PAGE and Transfer:

• Use 10-12% polyacrylamide gels for optimal resolution of the ~45 kDa STOML1 protein

• Transfer to PVDF membranes at 100V for 60-90 minutes in cold transfer buffer containing 20% methanol

Immunoblotting Protocol:

• Block membranes in 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Dilute primary STOML1 antibody 1:500-1:2000 in blocking buffer

• Incubate overnight at 4°C with gentle agitation

• Wash 3-5 times with TBST, 5-10 minutes each

• Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

• Wash 3-5 times with TBST, 5-10 minutes each

• Develop using ECL substrate and image

Validated Positive Controls:

• Human brain tissue lysates

• A375 cells

• Molt-4 cells

Expected Results:

• STOML1 should appear as a band at approximately 45 kDa

• Specificity can be confirmed by absence of the band in knockout samples or blocking with immunizing peptide

This protocol has been validated with multiple commercial STOML1 antibodies and provides consistent, specific detection of the target protein.

How should I design and interpret co-localization studies for STOML1 with other proteins?

For successful co-localization studies with STOML1:

Experimental Design:

• Antibody selection: Choose antibodies raised in different host species to allow simultaneous detection (e.g., rabbit anti-STOML1 with mouse anti-LAMP-2 for late endosome co-localization) .

• Confocal microscopy setup: Use appropriate filter sets with minimal spectral overlap and sequential scanning to prevent bleed-through. Capture z-stacks at Nyquist sampling rate to enable 3D reconstruction.

• Essential controls:

  • Single primary antibody with both secondary antibodies to check cross-reactivity

  • Secondary antibody-only controls to assess background

  • Peptide competition controls to verify specificity

Co-localization Analysis:

• Qualitative assessment: Look for punctate intracellular staining pattern for STOML1, particularly in perinuclear regions where late endosomes accumulate .

• Quantitative measures: Calculate Pearson's correlation coefficient and Manders' overlap coefficients. For STOML1 and stomatin, expected Pearson's values >0.7 indicate strong co-localization in late endosomal compartments .

• Biological validation: Confirm functional relevance by demonstrating that STOML1 overexpression causes redistribution of stomatin from plasma membrane to late endosomes .

Expected Patterns:

• STOML1 co-localizes with late endosomal markers like LAMP-1, LAMP-2, and Rab9

• STOML1 shows limited co-localization with early endosomal markers like Rab5

• In OSNs, STOML1 localizes to cell bodies but not the ciliary layer, unlike STOM which can be found in cilia

Interpretation Guidelines:

• Partial co-localization is expected since STOML1 may occupy subdomains within organelles

• Dynamic interactions may result in varying degrees of co-localization depending on cellular state

• Overexpression artifacts should be considered when using transfected cells

These guidelines ensure reliable and interpretable co-localization data for STOML1 studies.

What strategies can I use to study the functional implications of STOML1's interaction with ion channels?

To investigate STOML1's modulation of ion channels:

• Electrophysiological approaches:

  • Patch-clamp recordings in heterologous expression systems co-expressing STOML1 and acid-sensing ion channels (ASICs)

  • Focus on ASIC1 isoform 1 and ASIC3, as research shows STOML1 can specifically inhibit proton-gated current of ASIC1 and increase inactivation speed of ASIC3

  • Design experiments to measure changes in current amplitude, activation kinetics, and inactivation rates

• Structure-function analysis:

  • Create domain-specific mutations or deletions in STOML1 to identify regions critical for channel modulation

  • Focus on the stomatin domain, as this is likely involved in protein-protein interactions

  • Examine the role of the SCP-2 domain in modulating channel function through potential lipid environment alterations

• Calcium imaging:

  • Use fluorescent calcium indicators to monitor ASIC-mediated calcium influx in the presence or absence of STOML1

  • This approach allows higher throughput screening of STOML1 effects on channel function

• Biochemical interaction studies:

  • Investigate direct binding between STOML1 and ion channels using techniques like surface plasmon resonance

  • Use co-immunoprecipitation to confirm protein complex formation in native tissues

  • Employ proximity ligation assays to visualize interactions in situ

• Physiological models:

  • Study neuronal excitability in dorsal root ganglion neurons from wild-type versus STOML1 knockout animals

  • Examine proton-sensing mechanisms in these neurons, as STOML1 may be involved in their regulation

  • Assess behavioral responses to acidic stimuli, which may be altered in knockout models

• Super-resolution microscopy:

  • Investigate nanoscale co-localization of STOML1 and ion channels in neuronal membranes

  • Examine potential clustering effects that might influence channel function

These complementary approaches provide a comprehensive understanding of how STOML1 modulates ion channel function and the physiological consequences of these interactions.

How can I effectively study STOML1's role in cholesterol trafficking and lipid transfer?

To investigate STOML1's function in lipid trafficking:

• Cholesterol transport assays:

  • Use U18666A drug to block cholesterol efflux from late endosomes, then examine STOML1's effect on cholesterol accumulation

  • Fluorescent cholesterol analogs (e.g., BODIPY-cholesterol) can track trafficking in live cells

  • Filipin staining quantifies free cholesterol accumulation in fixed cells

• Lipid transfer assays:

  • Reconstitute purified STOML1 SCP-2 domain with donor and acceptor vesicles containing fluorescent lipids

  • Measure transfer rates between membranes in the presence/absence of STOML1

  • Compare wild-type STOML1 with SCP-2 domain mutants

• Subcellular fractionation:

  • Isolate late endosomal fractions and analyze their lipid composition

  • Compare lipid profiles between wild-type and STOML1-deficient cells

  • Focus on cholesterol and sphingolipid content differences

• Visualization techniques:

  • Induce STOML1 expression and look for formation of enlarged, cholesterol-filled, weakly LAMP-2-positive, acidic vesicles in the perinuclear region

  • This accumulation depends on the SCP-2 domain, suggesting its role in cholesterol transfer

• Domain-specific mutations:

  • Create STOML1 constructs lacking the SCP-2 domain

  • Compare these to full-length STOML1 in cholesterol trafficking assays

  • Research shows that cholesterol accumulation clearly depends on the SCP-2 domain

• Co-immunoprecipitation with lipid transfer proteins:

  • Identify potential interactions between STOML1 and other lipid transfer proteins

  • This may reveal cooperative mechanisms in cellular lipid distribution

• Lipidomic analysis:

  • Perform mass spectrometry-based lipidomics on cells overexpressing or lacking STOML1

  • Identify specific lipid species affected by STOML1 activity

These methods provide complementary approaches to elucidate STOML1's role in cholesterol trafficking and lipid transfer, particularly focusing on its unique SCP-2 domain function.

How can I utilize STOML1 antibodies in single-cell analysis techniques?

For incorporating STOML1 antibodies into single-cell analyses:

• Single-cell immunofluorescence approaches:

  • Use tyramide signal amplification methods to enhance detection sensitivity of low-abundance STOML1

  • Combine with high-content imaging systems for quantitative analysis of expression levels and subcellular localization

  • Implement machine learning algorithms to classify cells based on STOML1 expression patterns

• Flow cytometry applications:

  • Utilize conjugated STOML1 antibodies (FITC or HRP-conjugated variants are available)

  • Perform intracellular staining after membrane permeabilization, as STOML1 is primarily intracellular

  • Combine with markers of neuronal subtypes to identify specific cell populations expressing STOML1

• Mass cytometry (CyTOF):

  • Label STOML1 antibodies with rare earth metals

  • Combine with other metal-labeled antibodies to create high-dimensional profiles of STOML1-expressing cells

  • This approach allows simultaneous measurement of dozens of parameters in single cells

• Single-cell Western blotting:

  • Separate proteins from individual cells and probe for STOML1

  • Compare expression levels across heterogeneous cell populations

  • Particularly useful for rare cell types where bulk analysis might mask important differences

• Proximity extension assays:

  • Use oligonucleotide-labeled STOML1 antibodies for ultrasensitive detection

  • This method can detect protein expression in very small samples, even down to single cells

These techniques enable researchers to move beyond population averages and understand STOML1 function at the individual cell level, revealing heterogeneity that may be functionally significant, particularly in neuronal populations where STOML1 is highly expressed.

What are the emerging research directions for STOML1 antibodies in neurodegenerative disease studies?

Emerging applications of STOML1 antibodies in neurodegenerative research include:

• Biomarker development:

  • Investigate STOML1 as a potential biomarker in neurodegenerative conditions

  • STOML1's highest expression in brain tissue makes it relevant for neurological disorders

  • Antibody-based assays could quantify STOML1 in cerebrospinal fluid or brain-derived exosomes

• Cholesterol dysregulation in neurodegeneration:

  • Use STOML1 antibodies to study abnormal cholesterol accumulation in late endosomes

  • This is relevant to Niemann-Pick Type C and other lipid storage disorders

  • STOML1's role in cholesterol transfer to late endosomes makes it a potential target

• Protein-protein interaction networks:

  • Map STOML1 interactions with proteins implicated in neurodegeneration

  • Of particular interest is STOML1's interaction with FBXW7 isoform 3, which it may protect from degradation

  • FBXW7 is a tumor suppressor gene involved in ubiquitin-mediated proteolysis

• Ion channel modulation in neuropathic pain:

  • Investigate STOML1's modulation of acid-sensing ion channels in pain models

  • STOML1 inhibits proton-gated current of ASIC1 and increases inactivation speed of ASIC3

  • This may be relevant for developing novel analgesic approaches

• Neuroinflammatory responses:

  • Examine STOML1 expression in microglia and astrocytes during neuroinflammation

  • Antibodies could reveal alterations in expression or localization under pathological conditions

• Lysosomal function studies:

  • Investigate STOML1's relationship with lysosomal proteins in the context of lysosomal storage disorders

  • STOML1's late endosomal localization places it at the intersection of endocytic and lysosomal pathways