STOM Antibody

What is STOM protein and why is it significant in cell biology research?

STOM, also known as stomatin-like protein 2, is an integral membrane protein with 288 amino acid residues that plays essential roles in lipid raft organization and various cellular processes. The significance of STOM lies in its regulatory functions for ion channels and maintenance of cell membrane integrity. It participates in critical cellular processes including cell migration and adhesion .

STOM research has particular relevance for understanding diseases related to membrane defects and ion channel dysregulation. The protein's membrane localization and interaction with the cytoskeleton make it an important target for studies of cellular architecture and signaling pathways. Researchers investigating membrane biology, ion channel regulation, and related pathologies frequently utilize STOM as a model protein for understanding broader membrane protein dynamics .

What are the different types of STOM antibodies available for research purposes?

Researchers have access to several types of STOM antibodies, with polyclonal antibodies being among the most commonly used. Specific examples include:

• Polyclonal antibodies: Such as the STOM Rabbit Polyclonal Antibody (CAB6372), which recognizes human, mouse, and rat STOM and is validated for Western blot applications .

• Monoclonal antibodies: These offer higher specificity for particular epitopes of the STOM protein.

• Species-specific antibodies: Antibodies with reactivity to human, mouse, or rat STOM, with some offering cross-reactivity across multiple species .

The choice between these antibody types depends on the specific research application, with polyclonal antibodies offering broader epitope recognition and monoclonal antibodies providing higher specificity for particular regions of the protein.

How is specificity determined and validated for STOM antibodies?

Specificity validation for STOM antibodies typically involves multiple complementary approaches:

• Immunoblotting with positive control samples (mouse liver, spleen, heart, and rat liver for STOM antibodies)

• Comparison of detected molecular weight with the expected size (31.7 kDa for human STOM)

• Epitope mapping using recombinant protein fragments

• Testing in knockout/knockdown systems to confirm signal reduction

• Cross-validation using multiple antibodies targeting different epitopes

For example, the STOM Rabbit Polyclonal Antibody (CAB6372) is validated against recombinant fusion protein containing amino acids 55-288 of human STOM (NP_004090.4), which represents a significant portion of the full protein .

What applications are STOM antibodies validated for?

STOM antibodies are validated for several research applications, though the specific validations vary by product. Common applications include:

• Western blot (WB): The most common application, typically using dilutions between 1:500 and 1:2000

• ELISA: For quantitative detection of STOM in solution

• Immunohistochemistry (IHC): For detection in tissue sections

• Immunofluorescence (IF): For subcellular localization studies

• Immunoprecipitation (IP): For protein-protein interaction studies

Researchers should always verify the validation status for their specific application, as not all antibodies are validated for all possible techniques.

How should Western blot protocols be optimized for STOM detection?

Optimizing Western blot protocols for STOM detection requires attention to several key parameters:

• Sample preparation: For membrane proteins like STOM, complete solubilization is crucial. Use buffer systems containing 1-2% SDS or specialized membrane protein extraction buffers.

• Running conditions:

  • Use 10-12% polyacrylamide gels for optimal resolution of the 31.7 kDa STOM protein

  • Include positive control samples (mouse liver, spleen, heart, or rat liver)

• Transfer parameters:

  • For STOM, semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour is typically effective

  • PVDF membranes may provide better results than nitrocellulose for this membrane protein

• Antibody conditions:

  • Primary antibody dilution: Start with the manufacturer's recommended range (e.g., 1:500 - 1:2000)

  • Extended primary antibody incubation (overnight at 4°C) often improves detection

  • Include 0.05-0.1% Tween-20 in washing buffers to reduce background

• Detection system:

  • Enhanced chemiluminescence (ECL) systems work well for STOM detection

  • For quantitative analysis, consider fluorescence-based detection systems

What controls are essential when conducting experiments with STOM antibodies?

Proper experimental design with STOM antibodies requires several controls:

• Positive tissue controls: Mouse liver, spleen, heart, and rat liver are recommended positive controls for STOM antibody validation .

• Negative controls:

  • Primary antibody omission

  • Isotype control (non-specific IgG from the same species)

  • Ideally, STOM-knockout or knockdown samples

• Loading controls: For Western blot analysis, include appropriate loading controls:

  • Pan-membrane protein markers (e.g., Na⁺/K⁺-ATPase) for membrane fraction studies

  • Standard housekeeping genes (GAPDH, β-actin) for whole-cell lysates

• Peptide competition: Pre-incubation of the antibody with excess immunizing peptide should abolish specific signal.

• Multiple antibody validation: When possible, confirm findings using a second antibody targeting a different epitope of STOM.

How can I quantify STOM expression levels accurately?

Accurate quantification of STOM expression requires:

• Proper normalization strategies:

  • For Western blots: Normalize to appropriate loading controls

  • For qPCR analysis of STOM mRNA: Use multiple reference genes (GAPDH, β-actin, 18S rRNA)

• Standard curves:

  • For absolute quantification, generate standard curves using recombinant STOM protein

  • Ensure standards span the expected range of expression in samples

• Image analysis for Western blots:

  • Use software that corrects for background and performs densitometry

  • Ensure signal is within the linear range of detection

  • Analyze multiple independent biological replicates

• Complementary approaches:

  • Validate protein expression findings with mRNA analysis

  • Consider flow cytometry for cell-by-cell quantification in heterogeneous populations

How can I resolve non-specific binding when using STOM antibodies?

Non-specific binding is a common challenge with antibodies, including those targeting STOM. Resolution strategies include:

• Optimization of blocking conditions:

  • Test different blocking agents (5% BSA, 5% non-fat milk, commercial blockers)

  • Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

• Antibody dilution adjustment:

  • Generally, start with the manufacturer's recommended range (1:500 - 1:2000 for Western blot)

  • Perform titration experiments to determine optimal concentration

• Buffer modifications:

  • Add 0.1-0.3% Triton X-100 or 0.05-0.1% Tween-20 to reduce hydrophobic interactions

  • Include 5% serum from the secondary antibody species to reduce background

• Washing protocol enhancement:

  • Increase number and duration of washes

  • Use higher salt concentration (up to 500 mM NaCl) in wash buffers

• Pre-adsorption:

  • For tissues with high endogenous biotin, use avidin/biotin blocking kits

  • Consider pre-adsorbing antibodies with tissues/cells from species with anticipated cross-reactivity

What are the key considerations for subcellular localization studies of STOM?

STOM is primarily localized to the cell membrane with some cytoplasmic presence . For accurate subcellular localization studies:

• Sample preparation considerations:

  • Fixation method significantly impacts membrane protein preservation

  • For STOM, 4% paraformaldehyde fixation for 10-15 minutes is often optimal

  • Permeabilization should be gentle (0.1% Triton X-100 for 5-10 minutes)

• Co-localization markers:

  • Include established markers for cell membrane (e.g., Na⁺/K⁺-ATPase)

  • For lipid raft studies, co-stain with cholera toxin B subunit or caveolin-1

• Imaging considerations:

  • Super-resolution microscopy may be required to resolve lipid raft localization

  • Z-stack acquisition ensures complete visualization of membrane-associated proteins

• Validation approaches:

  • Confirm localization using subcellular fractionation followed by Western blot

  • Use epitope-tagged STOM constructs as complementary approach

How do I interpret contradictory results when using different STOM antibodies?

Contradictory results between antibodies targeting the same protein can stem from several factors:

• Epitope differences:

  • Different antibodies recognize distinct epitopes that may be differentially accessible

  • The STOM Rabbit Polyclonal Antibody (CAB6372) targets amino acids 55-288 , but other antibodies may target different regions

• Resolution strategies:

  • Map the epitopes of each antibody relative to functional domains of STOM

  • Verify which epitopes might be masked in certain experimental conditions

  • Consider that post-translational modifications may affect epitope accessibility

• Validation approaches:

  • Use genetic approaches (knockout/knockdown) to confirm specificity

  • Test antibodies in multiple applications to build confidence

  • Consider that different results may reflect biological reality (variant forms, modifications)

• Reporting recommendations:

  • Document all antibodies used (catalog numbers, lots, dilutions)

  • Report all results, even contradictory ones, with potential explanations

  • Consider publishing validation data alongside main findings

How can STOM antibodies be used to investigate protein-protein interactions?

STOM antibodies can be valuable tools for studying protein-protein interactions through several approaches:

• Co-immunoprecipitation (Co-IP):

  • Use STOM antibodies to pull down STOM and associated proteins

  • Analyze precipitated complexes by mass spectrometry or immunoblotting

  • Validation requires reversed Co-IP with antibodies against putative binding partners

• Proximity ligation assay (PLA):

  • Combines antibody recognition with DNA amplification to visualize interacting proteins

  • Requires antibodies raised in different species or directly conjugated to PLA probes

  • Offers single-molecule sensitivity and spatial resolution

• FRET/BRET applications:

  • For live-cell studies, combine STOM antibody fragments with fluorescent proteins

  • Enables real-time monitoring of dynamic interactions

  • Requires careful controls for fluorophore orientation effects

• Cross-linking followed by immunoprecipitation:

  • Chemical cross-linkers stabilize transient interactions before cell lysis

  • STOM antibodies then isolate STOM-containing complexes

  • Mass spectrometry identifies cross-linked partners

What are emerging applications of STOM antibodies in disease research?

STOM antibodies are increasingly utilized in disease-related research contexts:

• Membrane disorder studies:

  • STOM antibodies help investigate diseases associated with membrane defects

  • Quantification of STOM expression serves as a biomarker in certain conditions

  • Changes in STOM localization may indicate pathological alterations in membrane organization

• Ion channel dysfunction research:

  • STOM's role in regulating ion channels makes it relevant to channelopathies

  • Antibodies help correlate STOM expression/localization with channel function

  • Combined electrophysiology and immunolabeling approaches provide functional insights

• Cancer biology applications:

  • Altered STOM expression has been observed in certain cancers

  • Antibodies enable tissue microarray analysis across tumor types

  • Correlation of expression with clinical outcomes helps establish prognostic relevance

• Neurodegenerative disease research:

  • Membrane protein dysfunction is implicated in several neurodegenerative conditions

  • STOM antibodies facilitate investigation of membrane integrity in affected tissues

  • Post-mortem tissue analysis requires specialized protocols for degradation-sensitive epitopes

How do experimental approaches differ when working with STOM in different species?

Working with STOM across species requires consideration of several factors:

How might advances in antibody engineering improve STOM-targeted research tools?

Emerging antibody engineering approaches offer several potential improvements for STOM research:

• Single-domain antibodies (nanobodies):

  • Smaller size allows better access to sterically hindered epitopes

  • Potential for improved penetration in tissue sections

  • Greater stability for long-term storage and challenging conditions

• Recombinant antibody fragments:

  • Fab or scFv formats eliminate Fc-mediated artifacts

  • Production in bacterial systems reduces batch-to-batch variability

  • Site-specific conjugation enables precise positioning of labels

• Bispecific antibodies:

  • Simultaneous binding to STOM and another protein of interest

  • Enables novel proximity-based detection strategies

  • May improve sensitivity for co-localization studies

• Engineering for subcellular targeting:

  • Membrane-permeable antibodies for live-cell applications

  • Compartment-specific targeting sequences for organelle studies

  • pH-responsive antibodies for endosomal tracking

What methodological advances are improving antibody validation for STOM research?

Recent developments in antibody validation are enhancing confidence in STOM antibody research:

• CRISPR/Cas9 knockout validation:

  • Generation of STOM-knockout cell lines for definitive specificity testing

  • Provides gold-standard negative controls for antibody validation

  • Allows identification of potential cross-reactivity with related proteins

• Multi-omic correlation approaches:

  • Correlation of antibody-based detection with RNA-seq and proteomics data

  • Triangulation increases confidence in expression patterns

  • Helps identify discrepancies that may indicate technical issues

• Structural validation:

  • Epitope mapping using hydrogen-deuterium exchange mass spectrometry

  • Computational prediction of epitope accessibility in different conformational states

  • Cryo-EM validation of antibody binding to target structure

• Community-based validation resources:

  • Antibody validation databases with user-contributed data

  • Standardized reporting formats for validation experiments

  • Pre-competitive collaborations among research institutions