TM4SF20 Antibody

What is TM4SF20 and what cellular functions does it perform?

TM4SF20 (Transmembrane 4 L6 Family Member 20) is a member of the 4-transmembrane L6 superfamily that encodes a surface protein containing four transmembrane domains . This protein is primarily expressed in the adult mammalian brain, with notable presence in the parietal and occipital lobes, hippocampus, pons, white matter, corpus callosum, and cerebellum .

Functionally, TM4SF20 interacts with integrins to mediate cell adhesion, proliferation, and motility processes . The protein has also been implicated in promoting angiogenic activities in endothelial cells through vascular endothelial growth factor (VEGF) induction . Recent genetic studies have identified TM4SF20 as potentially significant in neurodevelopment, as deletions in this gene have been associated with language delay and white matter abnormalities in pediatric populations .

When designing experiments to investigate TM4SF20 function, researchers should consider its tissue-specific expression patterns and potential roles in both normal development and pathological conditions.

How should TM4SF20 antibodies be validated for experimental use?

Validation of TM4SF20 antibodies should follow a multi-step approach to ensure specificity and reliability in experimental applications:

• Epitope verification: Confirm that the antibody recognizes the intended region of TM4SF20. The antibody described in the search results targets amino acids 114-184 of the human TM4SF20 protein .

• Western blot analysis: Perform western blotting using positive control samples known to express TM4SF20 and negative controls. Look for bands of the expected molecular weight.

• Cross-reactivity testing: Test the antibody against similar proteins, particularly other members of the transmembrane 4 L6 family, to ensure specificity.

• Knockout/knockdown validation: Use samples from knockout models or siRNA-treated cells to confirm antibody specificity.

• Application-specific validation: For each application (IHC, ICC, ELISA), specific validation protocols should be employed to verify that the antibody performs consistently under the conditions of that particular technique.

TM4SF20 antibodies with demonstrated reactivity to human samples should be validated at the recommended dilutions (IHC: 1/20-1/200, IF/ICC: 1/50-1/200) .

What are the optimal storage conditions for maintaining TM4SF20 antibody activity?

To maintain optimal activity of TM4SF20 antibodies, follow these evidence-based storage recommendations:

• Temperature conditions: Store antibody aliquots at -20°C for long-term preservation . Avoid keeping antibodies at room temperature for extended periods.

• Aliquoting strategy: Upon receipt, divide the antibody solution into multiple small aliquots before freezing to minimize freeze-thaw cycles .

• Freeze-thaw considerations: Repeated freeze-thaw cycles significantly reduce antibody activity and specificity. Limit these cycles by using small working aliquots .

• Buffer composition: TM4SF20 antibodies are typically supplied in a buffer containing 0.01 M PBS (pH 7.4), 0.03% Proclin-300, and 50% Glycerol . This formulation helps maintain stability during storage.

• Working solution handling: When using the antibody, keep the working solution on ice during experiment preparation.

• Quality control: Periodically test stored antibodies against fresh standards to ensure they maintain reactivity and specificity.

Proper storage is critical for maintaining the >95% purity level of research-grade TM4SF20 antibodies .

How can TM4SF20 antibodies be used to study the cellular mislocalization caused by TM4SF20 exon 3 deletion?

The TM4SF20 exon 3 deletion results in a truncated protein that mislocalizes to the cytoplasm rather than properly targeting to the plasma membrane . To investigate this phenomenon:

• Comparative immunocytochemistry protocol:

  • Culture appropriate cell lines (such as neuroblastoma Neuro-2a cells)

  • Transfect cells with constructs expressing either wild-type or truncated TM4SF20

  • Fix cells with 4% paraformaldehyde (20 minutes, room temperature)

  • Permeabilize with 0.1% Triton X-100 (10 minutes)

  • Block with 5% BSA (1 hour)

  • Incubate with TM4SF20 antibody at 1:100 dilution (overnight, 4°C)

  • Apply fluorescent secondary antibody (1 hour, room temperature)

  • Counterstain nuclei with DAPI

  • Image using confocal microscopy

• Analysis parameters: Score at least 50 transfected cells per condition in duplicate experiments to quantify localization patterns . Wild-type TM4SF20 should predominantly localize to the cell membrane, while the truncated form will accumulate in the cytoplasm.

• Controls: Include untransfected cells and secondary-antibody-only controls. When possible, use cells from patients with the TM4SF20 deletion for physiological relevance.

This approach has successfully demonstrated the differential localization of wild-type versus truncated TM4SF20 in previous studies .

What considerations should be made when using TM4SF20 antibodies in brain tissue analyses?

When applying TM4SF20 antibodies to brain tissue analyses, researchers should consider several specialized factors:

• Tissue preparation:

  • Fixation method significantly impacts epitope preservation; paraformaldehyde fixation followed by sucrose cryoprotection is recommended for immunohistochemical detection

  • For frozen sections, optimal thickness is 5-10 μm

  • For paraffin sections, antigen retrieval is critical (citrate buffer pH 6.0, 20 minutes)

• Regional expression patterns: Target analyses to brain regions with known TM4SF20 expression, including parietal lobe, occipital lobe, hippocampus, pons, white matter, corpus callosum, and cerebellum .

• Cell-type specific considerations: Use dual-labeling with cell-type specific markers (neurons, astrocytes, oligodendrocytes) to determine the cellular distribution of TM4SF20 within brain tissues.

• White matter studies: Given the association between TM4SF20 deletion and white matter hyperintensities, special attention should be paid to white matter regions :

  • Use TM4SF20 antibodies in conjunction with myelin markers

  • Compare punctate and multifocal patterns in periventricular and deep white matter

  • Consider age-dependent expression patterns, especially in pediatric samples

• Dilution optimization: Typically start with 1:100 dilution and adjust based on signal intensity and background .

• Blocking strategy: Use 5-10% serum from the same species as the secondary antibody plus 1% BSA to minimize background staining.

How can TM4SF20 antibodies be integrated into studies of neurodevelopmental disorders?

TM4SF20 antibodies offer valuable tools for investigating neurodevelopmental disorders, particularly those involving language delay and white matter abnormalities:

• Case-control immunohistochemical studies:

  • Compare TM4SF20 protein expression and localization in post-mortem brain tissue from patients with language disorders versus controls

  • Focus on regions implicated in language processing and white matter tracts

  • Quantify expression differences using standardized image analysis techniques

• Developmental expression profiling:

  • Use TM4SF20 antibodies to track protein expression throughout neurodevelopment in animal models

  • Correlate expression patterns with critical developmental windows for language acquisition

  • Examine potential differences in TM4SF20 localization during myelination processes

• Integration with neuroimaging:

  • Combine immunohistochemical findings with MRI data to correlate TM4SF20 expression with white matter hyperintensities

  • Design studies that connect molecular data with the T2 hyperintensities observed in individuals with TM4SF20 deletion

• Genetic variant analysis:

  • Use TM4SF20 antibodies to assess the functional consequences of the complex 4 kb deletion in 2q36.3 and other variants

  • Compare protein expression and localization between samples from different ethnic backgrounds, particularly focusing on Southeast Asian populations where the deletion has higher prevalence

• Protocol considerations:

  • For developmental studies, use consistent fixation and processing methods across age points

  • Apply antibody at 1:50-1:100 dilution for maximal sensitivity in pediatric samples

  • Include appropriate age-matched controls

What methodologies are recommended for studying TM4SF20 interactions with integrins?

TM4SF20 has been shown to interact with integrins to mediate cell adhesion, proliferation, and motility . To investigate these interactions:

• Co-immunoprecipitation protocol:

  • Lyse cells in non-denaturing buffer (1% NP-40, 150mM NaCl, 50mM Tris pH 7.4)

  • Pre-clear lysate with Protein G beads

  • Incubate cleared lysate with TM4SF20 antibody (2-5 μg per mg protein)

  • Capture complexes with Protein G beads

  • Wash extensively with lysis buffer

  • Elute and analyze by Western blot for integrin partners

• Proximity ligation assay (PLA):

  • Fix cells with 4% paraformaldehyde

  • Permeabilize and block as standard

  • Incubate with TM4SF20 antibody and antibody against suspected integrin partner

  • Follow PLA protocol with appropriate PLA probes

  • Visualize interaction signals by fluorescence microscopy

• FRET/FLIM analysis:

  • Label TM4SF20 antibody with donor fluorophore

  • Label integrin antibody with acceptor fluorophore

  • Perform FRET measurements in fixed cells or tissue sections

  • Calculate FRET efficiency as indicator of protein proximity

• Functional blocking studies:

  • Treat cells with TM4SF20 antibody to potentially disrupt interactions with integrins

  • Assess functional outcomes such as cell adhesion, migration, or proliferation

  • Compare with integrin-blocking antibodies to determine specificity

• Angiogenesis assays:

  • Given TM4SF20's role in promoting angiogenic activities through VEGF induction , design experiments with endothelial cells

  • Use TM4SF20 antibodies in tube formation assays

  • Assess impact on VEGF expression and integrin-mediated endothelial cell behaviors

How should research protocols be modified when detecting truncated versus wild-type TM4SF20?

The TM4SF20 deletion results in a truncated protein missing two of its four transmembrane domains . To effectively detect and distinguish between wild-type and truncated TM4SF20:

• Epitope selection considerations:

  • Confirm whether your TM4SF20 antibody's epitope (amino acids 114-184) is present in both wild-type and truncated forms

  • For detection of both forms, choose antibodies targeting regions preserved in the truncated protein

  • For specific detection of the full-length protein, select antibodies against regions deleted in the truncated form

• Western blot modifications:

  • Use gradient gels (4-20%) to better resolve the size difference

  • Extended run times may be necessary to clearly separate wild-type (~20 kDa) from truncated protein

  • Include positive controls expressing each form

• Immunofluorescence protocol adjustments:

  • Increase antibody concentration (1:50 dilution) when detecting the truncated protein

  • Use confocal microscopy with z-stack analysis to distinguish membrane from cytoplasmic localization

  • Co-stain with membrane markers to confirm localization patterns

• Flow cytometry considerations:

  • For detection of cell-surface TM4SF20, use non-permeabilized cells

  • For detection of mislocalized cytoplasmic protein, use permeabilized cells

  • Compare surface:intracellular ratios between wild-type and cells expressing the truncated form

• Minigene assay approach:

  • To study the splicing consequences of the deletion, implement the minigene assay as described in previous research

  • Generate constructs spanning exon 2 through exon 4

  • Transfect cells and analyze splicing products by RT-PCR

What are common challenges when working with TM4SF20 antibodies and how can they be addressed?

Researchers working with TM4SF20 antibodies may encounter several technical challenges. Here are evidence-based solutions:

• High background in immunostaining:

  • Increase blocking time (2-3 hours) and concentration (10% serum)

  • Include 0.1-0.3% Triton X-100 in blocking solution to reduce non-specific binding

  • Optimize antibody dilution; start with recommended range (1:20-1:200 for IHC; 1:50-1:200 for IF/ICC)

  • Include 0.1% BSA in antibody dilution buffer

  • Increase washing steps (5 × 5 minutes)

• Weak or absent signal:

  • Verify target expression in your sample type

  • Perform antigen retrieval (for IHC): citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Increase antibody concentration within recommended range

  • Extend primary antibody incubation (overnight at 4°C)

  • Use amplification systems (e.g., biotin-streptavidin)

  • Ensure antibody storage conditions are optimal (-20°C, avoid freeze-thaw cycles)

• Non-specific bands on Western blot:

  • Increase blocking time and stringency

  • Optimize antibody dilution

  • Include controls: lysates from cells with known TM4SF20 expression levels

  • Use gradient gels for better resolution

  • Consider more stringent washing conditions

• Inconsistent results between experiments:

  • Standardize sample collection and processing

  • Prepare master mixes for antibody dilutions

  • Use consistent incubation times and temperatures

  • Document all protocol variables

  • Include positive controls in each experiment

• Detection issues in clinical samples:

  • Consider fixation methods; formalin-fixed samples may require extended antigen retrieval

  • For tissues from Southeast Asian populations with potential TM4SF20 deletion , ensure antibody epitope is not in the deleted region

  • Compare results with TM4SF20 mRNA detection methods

How can TM4SF20 antibody performance be optimized for studying white matter hyperintensities?

To optimize TM4SF20 antibody performance specifically for studying white matter hyperintensities (WMHs) associated with TM4SF20 deletion:

• Sample preparation protocol modifications:

  • For fresh-frozen brain tissue sections: fix in 4% PFA for 10 minutes, then proceed with standard immunohistochemistry

  • For paraffin-embedded sections: extended antigen retrieval may be necessary (20-30 minutes in citrate buffer)

  • Consider vibratome sectioning (50-100 μm) for 3D analysis of white matter regions

• Co-staining strategy:

  • Combine TM4SF20 antibody with markers for:

    • Oligodendrocytes (Olig2, CNPase)

    • Myelin (MBP, PLP)

    • Astrocytes (GFAP)

    • Microglia (Iba1)

  • Use sequential immunostaining to avoid cross-reactivity

• Imaging approach:

  • Use confocal microscopy with z-stack capabilities for high-resolution 3D imaging

  • Employ tile scanning for large-area white matter analysis

  • Standardize exposure settings between control and WMH samples

  • Collect quantitative measures: intensity, colocalization coefficients, morphological parameters

• Control tissue selection:

  • Include age-matched control samples without WMHs

  • Consider analyzing samples from individuals with WMHs not associated with TM4SF20 deletion

  • Where possible, include samples from Southeast Asian populations where the TM4SF20 deletion is enriched

• Correlation with MRI data:

  • Register immunohistochemistry sections with MRI images when possible

  • Focus analysis on periventricular and deep white matter regions where T2 hyperintensities are typically observed

  • Quantify TM4SF20 expression in relation to WMH severity

What novel applications of TM4SF20 antibodies are emerging in neurodevelopmental research?

Emerging applications of TM4SF20 antibodies in neurodevelopmental research include:

• Single-cell protein analysis:

  • Adapting TM4SF20 antibodies for mass cytometry (CyTOF) to analyze protein expression at single-cell resolution

  • Combining with other markers to create comprehensive cellular atlases of developing brain regions

  • Quantifying cell-to-cell variability in expression patterns

• In vivo imaging approaches:

  • Developing near-infrared-labeled TM4SF20 antibodies for deep tissue imaging

  • Creating antibody-based probes for PET imaging to visualize TM4SF20 expression in living subjects

  • Correlating molecular imaging with structural and functional neuroimaging

• Therapeutic targeting:

  • Exploring antibody-based approaches to modulate TM4SF20 function

  • Investigating whether blocking mislocalized TM4SF20 could ameliorate associated phenotypes

  • Developing strategies to enhance proper TM4SF20 trafficking in deletion carriers

• Population-specific biomarker development:

  • Utilizing TM4SF20 antibodies to develop diagnostic tools for early identification of language delay risk

  • Creating screening assays particularly relevant for Southeast Asian populations where the deletion is enriched (approximately 1% allele frequency in Vietnamese Kinh individuals)

  • Establishing prognostic indicators based on TM4SF20 expression patterns

• Developmental timing studies:

  • Mapping TM4SF20 expression throughout neurodevelopment

  • Identifying critical periods where altered expression may have maximal impact

  • Correlating with white matter development milestones and language acquisition windows

How can TM4SF20 antibodies contribute to understanding the mechanism of language delay in affected individuals?

TM4SF20 antibodies can provide crucial insights into the mechanisms underlying language delay in affected individuals through several research approaches:

• Circuit-specific analysis:

  • Use TM4SF20 antibodies to map protein expression in language-related neural circuits

  • Compare expression patterns between typical development and cases with TM4SF20 deletion

  • Focus on left perisylvian language areas and associated white matter tracts

• Cellular pathology investigation:

  • Analyze how mislocalized TM4SF20 affects cellular morphology in neurons and glia

  • Examine potential downstream effects on cell adhesion molecules and synaptic proteins

  • Investigate consequences for neuronal migration and connectivity

• Functional correlation studies:

  • Correlate TM4SF20 expression patterns with language assessment scores in clinical cohorts

  • Design age-appropriate language measures for pediatric populations

  • Create standardized protocols for combined protein expression and functional outcomes

• Developmental trajectory mapping:

  • Track TM4SF20 expression throughout critical periods of language development

  • Compare with developmental milestones in language acquisition

  • Identify potential intervention windows based on protein expression patterns

• Translational models:

  • Develop organoid models expressing wild-type or truncated TM4SF20

  • Use antibodies to characterize protein expression and localization in these models

  • Correlate with electrophysiological measures of neuronal activity and connectivity

The combination of these approaches, centered on careful application of TM4SF20 antibodies, can significantly advance our understanding of how TM4SF20 deletion contributes to language delay, particularly in the Southeast Asian populations where this genetic variant shows enrichment .