TMEM222 Antibody

What is TMEM222 and what biological functions has research identified?

TMEM222 (Transmembrane Protein 222) is a 208-amino acid protein belonging to the heterogeneous group of transmembrane proteins that span lipid bilayers of various cell membranes. The protein contains three transmembrane domains and a domain of unknown function (DUF778) . Recent research has identified TMEM222's potential role in brain development and function, as biallelic variants in the TMEM222 gene were discovered in patients with autosomal recessive intellectual disability . While its precise molecular function remains under investigation, subcellular localization studies have revealed that TMEM222 localizes to early endosomes in synapses of mature neurons derived from induced pluripotent stem cells (iPSCs) . This localization pattern suggests potential involvement in synaptic function, possibly through endosomal trafficking mechanisms that are critical for neuronal development and signaling.

In which human tissues is TMEM222 predominantly expressed?

Expression analysis demonstrates that TMEM222 has relatively high expression levels in the human brain, with particularly notable expression in the parietal and occipital cortex . Researchers have conducted quantitative reverse transcription polymerase chain reaction (RT-qPCR) across 13 brain areas, 9 fetal tissues, and 10 adult tissues to characterize the expression profile of TMEM222 . This expression pattern aligns with the neurodevelopmental phenotypes observed in patients with TMEM222 mutations, further supporting its critical role in brain function and development. The expression data provides valuable information for researchers designing experiments to investigate TMEM222's function in specific brain regions.

What validated applications exist for TMEM222 antibodies in research?

TMEM222 antibodies have been validated for several standard immunological applications in research settings. These include Enzyme-Linked Immunosorbent Assay (ELISA), Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF) . Commercially available antibodies, such as the TMEM222 Polyclonal Antibody, have been specifically tested and validated for detecting human TMEM222 protein in these applications . When selecting antibodies for experimental use, researchers should verify the validation data for their specific application and carefully review the antibody specifications, including the immunogen used for antibody production, to ensure compatibility with their experimental system.

How can researchers use TMEM222 antibodies to investigate neurodevelopmental disorders?

TMEM222 antibodies represent valuable tools for investigating the molecular pathology underlying TMEM222-associated neurodevelopmental disorders. Researchers can employ these antibodies in comparative studies between patient-derived and control samples using the following approaches:

• Protein expression analysis: Western blotting with TMEM222 antibodies can reveal alterations in protein expression levels or unexpected molecular weight patterns that might result from pathogenic variants.

• Subcellular localization studies: Immunofluorescence using TMEM222 antibodies in neuronal cultures can determine whether disease-causing variants affect the normal localization of TMEM222 to early endosomes in synapses .

• Brain tissue studies: Immunohistochemistry on post-mortem brain sections can map TMEM222 distribution across different cell types and brain regions, potentially revealing alterations in patients with neurodevelopmental disorders.

• Patient-derived iPSC models: Generating iPSC-derived neurons from patients with TMEM222 variants allows for detailed investigation of protein expression, localization, and function in a disease-relevant context .

These approaches can provide mechanistic insights into how TMEM222 mutations lead to intellectual disability and associated phenotypes, potentially identifying therapeutic targets for intervention.

What experimental controls are critical when using TMEM222 antibodies?

When working with TMEM222 antibodies, implementing proper controls is essential for generating reliable and interpretable results:

For genetic studies involving TMEM222, appropriate wild-type controls alongside variant-expressing constructs are essential when assessing functional consequences of specific mutations identified in patients .

What methods can accurately assess TMEM222 protein interactions and complexes?

Investigating TMEM222 protein interactions requires specialized techniques suitable for membrane proteins:

• Co-immunoprecipitation with membrane protein adaptations:

  • Use gentle detergents (e.g., digitonin, DDM) that preserve membrane protein complexes

  • Consider crosslinking approaches to stabilize transient interactions

  • Employ TMEM222 antibodies for pull-down experiments followed by mass spectrometry

• Proximity labeling approaches:

  • BioID or APEX2 fusion constructs with TMEM222 can identify proximal proteins in living cells

  • These methods are particularly valuable for transmembrane proteins like TMEM222 that reside in endosomal compartments

• Fluorescence-based interaction studies:

  • FRET (Förster Resonance Energy Transfer) between TMEM222 and candidate interacting proteins

  • Bimolecular Fluorescence Complementation (BiFC) to visualize interactions in cellular contexts

• Yeast two-hybrid membrane adaptations:

  • Split-ubiquitin membrane yeast two-hybrid systems designed specifically for membrane proteins

  • Allows screening for interactions between TMEM222 and other membrane or cytosolic proteins

When designing these experiments, researchers should consider TMEM222's localization to early endosomes in synapses and focus on potential interactions with endosomal trafficking machinery that might explain its role in neurodevelopment.

What is the optimal protocol for Western blot detection of TMEM222?

For reliable Western blot detection of TMEM222, researchers should consider this optimized protocol:

Sample Preparation:

• Extract proteins using a membrane protein-compatible lysis buffer containing:

  • Standard lysis buffer with protease inhibitor cocktail

  • 1% NP-40 or 0.5% Triton X-100

  • Avoid boiling samples to prevent membrane protein aggregation; instead incubate at 37°C for 30 minutes

Gel Electrophoresis and Transfer:

• Use 12-15% SDS-PAGE gels to properly resolve the 208-amino acid TMEM222 protein

• Transfer to nitrocellulose membrane using standard wet transfer at 30V overnight at 4°C for optimal transfer of membrane proteins

Antibody Incubation and Detection:

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

• Incubate with anti-TMEM222 antibody at 1:2000 dilution overnight at 4°C

• Wash 3×10 minutes with TBST

• Incubate with HRP-conjugated or IRDye-labeled secondary antibody (1:10,000)

• Develop using enhanced chemiluminescence or fluorescent scanning

Expected Results:

• TMEM222 should appear at approximately 23-25 kDa

• Use positive controls such as HEK293T cells transfected with wild-type TMEM222 constructs

• Internal loading control: anti-tubulin (1:2,000)

This protocol incorporates modifications specifically for transmembrane proteins and has been used successfully in published research on TMEM222 .

How can researchers optimize immunofluorescence protocols for TMEM222 in neuronal cultures?

For optimal detection of TMEM222 in neuronal cultures, especially iPSC-derived neurons, follow this specialized protocol:

Cell Culture and Fixation:

• Culture neurons on poly-L-lysine coated coverslips for 4 weeks to ensure mature synaptic development

• Fix cells with 4% paraformaldehyde in PBS for 15 minutes at room temperature

• Consider using a gentler fixative (2% paraformaldehyde) for better preservation of membrane structures

Permeabilization and Blocking:

• Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes

• Block with 10% normal goat serum and 1% BSA in PBS for 1 hour at room temperature

Antibody Incubation:

• Incubate with anti-TMEM222 antibody (1:250-1:500) overnight at 4°C

• Co-stain with markers for:

  • Early endosomes (anti-EEA1) to confirm endosomal localization

  • Synaptic markers (anti-Synapsin) to identify synaptic regions

• Use appropriate secondary antibodies with minimal cross-reactivity (1:1000)

Imaging Considerations:

• Use confocal microscopy with Airyscan or similar super-resolution capabilities to resolve endosomal structures

• Capture Z-stacks (0.3μm step size) to fully visualize three-dimensional distribution

• For co-localization studies, ensure proper channel alignment and use appropriate co-localization metrics

This optimized protocol accounts for TMEM222's known localization to early endosomes in neuronal synapses and will help researchers accurately detect the protein in cellular contexts relevant to neurodevelopmental disorders.

What are recommended approaches for validating TMEM222 antibody specificity?

To rigorously validate TMEM222 antibody specificity, researchers should implement a multi-faceted approach:

• Genetic validation approaches:

  • Test antibody in CRISPR/Cas9 TMEM222 knockout cell lines

  • Use siRNA/shRNA knockdown models to confirm signal reduction

  • Examine signal in cells overexpressing tagged TMEM222 constructs

• Epitope mapping and competition assays:

  • Conduct peptide competition with the immunogen peptide (aa 77-164 for some commercial antibodies)

  • Test antibody against truncated TMEM222 constructs to confirm epitope recognition

• Multi-technique validation:

  • Cross-validate detection across Western blot, IF, and IHC

  • Confirm that antibody recognizes both native and denatured protein forms appropriately

  • Compare results from multiple different antibody clones targeting different TMEM222 epitopes

• Controls using patient samples:

  • If available, test antibody recognition in samples from individuals with biallelic TMEM222 loss-of-function variants

  • This provides physiologically relevant validation in disease-relevant models

• Mass spectrometry confirmation:

  • Perform immunoprecipitation followed by mass spectrometry to confirm antibody captures TMEM222

  • This approach provides unbiased confirmation of antibody target specificity

Implementing these validation steps ensures that experimental findings using TMEM222 antibodies are reliable and reproducible, particularly important when investigating a protein implicated in human neurodevelopmental disorders.

How should researchers design experiments to investigate TMEM222 variants identified in neurodevelopmental disorders?

Researchers investigating TMEM222 variants can implement this comprehensive experimental framework:

• Expression analysis of variant proteins:

  • Generate expression constructs containing identified variants (e.g., p.(Val179del), p.(Gly72Ser), p.(Gly176Arg))

  • Assess protein expression levels via Western blot to determine if variants affect protein stability

  • Examine subcellular localization using immunofluorescence to detect mislocalization

• Functional studies in neuronal models:

  • Use lentiviral expression systems for wild-type and variant TMEM222 in iPSC-derived neurons

  • Assess endosomal morphology and trafficking using live-cell imaging

  • Evaluate synaptic density and function using electrophysiology and synaptic markers

• Patient-derived cellular models:

  • Generate iPSCs from patients with TMEM222 variants and differentiate to neurons

  • Characterize neuronal development, morphology, and function

  • Perform rescue experiments with wild-type TMEM222 to confirm causality

• Molecular interaction studies:

  • Compare wild-type and variant TMEM222 interactomes using proximity labeling or co-immunoprecipitation

  • Focus on endosomal trafficking machinery interactions that might be disrupted by variants

• Validation in animal models:

  • Generate TMEM222 knockout or variant knock-in models in appropriate organisms

  • Characterize neurodevelopmental and behavioral phenotypes

This systematic approach enables comprehensive characterization of how specific TMEM222 variants contribute to neurodevelopmental phenotypes, potentially identifying intervention points for therapeutic development.

What common challenges might researchers encounter when working with TMEM222 antibodies and how can they be addressed?

Researchers working with TMEM222 antibodies may encounter several technical challenges that can be addressed through specific methodological adaptations:

By anticipating these challenges and implementing appropriate technical solutions, researchers can generate more reliable and consistent results when studying TMEM222 in various experimental systems.

What approaches can help resolve contradictory findings when studying TMEM222 localization or expression?

When faced with contradictory results regarding TMEM222 localization or expression, researchers should implement this systematic troubleshooting approach:

• Antibody validation and comparison:

  • Test multiple antibodies targeting different TMEM222 epitopes

  • Verify specificity through knockout/knockdown controls for each antibody

  • Compare polyclonal and monoclonal antibodies if available

• Multi-methodology confirmation:

  • Combine biochemical fractionation with immunological detection

  • Use orthogonal techniques:

    • Fluorescent protein tagging (N and C-terminal tags)

    • Proximity labeling approaches

    • Mass spectrometry of isolated subcellular compartments

• Cell type and context considerations:

  • Verify findings across multiple cell types and developmental stages

  • Remember that TMEM222 has highest expression in specific brain regions (parietal and occipital cortex)

  • Consider potential dynamic localization changes under different conditions

• Technical parameter examination:

  • Systematically compare fixation and permeabilization protocols

  • Evaluate sample preparation methods for potential artifacts

  • Consider protein extraction efficiency for different subcellular compartments

• Functional validation:

  • Design experiments that test function at the proposed localization site

  • Use domain-specific mutations to alter localization and assess consequences

By implementing this methodical approach, researchers can resolve contradictory findings and establish a consensus regarding TMEM222's authentic localization and expression patterns in physiologically relevant contexts.

How might TMEM222 antibodies be used to advance understanding of endosomal function in neurons?

TMEM222 antibodies can serve as valuable tools for investigating endosomal biology in neurons, particularly given TMEM222's localization to early endosomes in synapses . Researchers can leverage these antibodies in several cutting-edge approaches:

• High-resolution mapping of endosomal subdomains:

  • Use super-resolution microscopy with TMEM222 antibodies alongside markers for endosomal subdomains

  • Determine if TMEM222 occupies specific microdomains within early endosomes that might represent specialized functional zones

• Live trafficking studies:

  • Combine antibody fragments with quantum dots for single-particle tracking

  • Monitor TMEM222-positive endosome dynamics in response to neuronal activity

  • Correlate trafficking patterns with synaptic events and plasticity

• Proteomic analysis of TMEM222-positive endosomes:

  • Use TMEM222 antibodies for immunoisolation of endosomal compartments

  • Perform mass spectrometry to identify the comprehensive protein composition

  • Compare composition between normal and neurodevelopmental disorder models

• Functional modulation studies:

  • Apply TMEM222 antibodies to living neurons to potentially modulate protein function

  • Assess consequences on endosomal trafficking, recycling, and degradation pathways

  • Evaluate effects on synaptic transmission and plasticity

These approaches can reveal TMEM222's contribution to endosomal function in neurons and potentially identify novel therapeutic targets for neurodevelopmental disorders associated with endosomal dysfunction.

What role might TMEM222 play in neuronal development based on current evidence?

Based on available research, TMEM222 likely contributes to neuronal development through several potential mechanisms that warrant further investigation:

• Endosome-mediated receptor trafficking:

  • Given its localization to early endosomes , TMEM222 may regulate the trafficking of key neurodevelopmental receptors

  • This could affect signaling pathways critical for neuronal migration, differentiation, or synaptogenesis

  • Developmental timing of TMEM222 expression could reveal critical periods of function

• Synaptic vesicle cycling:

  • The presence of TMEM222 in synapses suggests potential roles in synaptic vesicle endocytosis or recycling

  • Disruption might alter neurotransmitter release or synaptic plasticity mechanisms

  • This could explain the intellectual disability phenotype observed in patients

• Membrane protein sorting:

  • TMEM222 might function in sorting machinery that directs proteins to axons versus dendrites

  • Variants could disrupt neuronal polarity or compartmentalization

  • This would align with the decreased muscle mass and motor symptoms observed in some patients

• Neuron-glia communication:

  • Endosomal proteins can mediate exchange of signals between neurons and supporting cells

  • TMEM222 dysfunction might disrupt these interactions, affecting myelination or synapse pruning

Future research using TMEM222 antibodies to track protein expression and localization throughout neuronal development will help clarify its specific contributions to neurodevelopmental processes and how variants lead to intellectual disability.

How can TMEM222 antibodies be optimized for use in human brain tissue sections?

For optimal detection of TMEM222 in human brain tissue sections, particularly from regions with high expression such as parietal and occipital cortex , researchers should implement this specialized protocol:

Tissue Preparation:

• Use either fresh-frozen tissue or formalin-fixed paraffin-embedded (FFPE) sections (10μm thickness)

• For FFPE sections, perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 20 minutes

• For fresh-frozen sections, fix briefly (10 minutes) with 4% paraformaldehyde post-sectioning

Blocking and Permeabilization:

• Block endogenous peroxidase activity with 0.3% H₂O₂ if using HRP detection

• Permeabilize with 0.2% Triton X-100 in PBS for 15 minutes

• Block with 10% normal serum from secondary antibody host species plus 1% BSA for 2 hours

Antibody Application:

• Apply TMEM222 antibody at 1:100-1:200 dilution in blocking buffer

• Incubate for 48-72 hours at 4°C to ensure penetration into tissue

• For co-localization studies, apply antibodies sequentially rather than simultaneously

• Include lipofuscin autofluorescence quencher (e.g., TrueBlack) before mounting

Signal Development:

• For chromogenic detection, use DAB with nickel enhancement for improved sensitivity

• For fluorescence, use tyramide signal amplification to enhance detection of low-abundance proteins

• Counterstain with DAPI and include markers for neurons (NeuN) and endosomes (EEA1)

This tailored approach accounts for the specific challenges of detecting transmembrane proteins in complex brain tissue and will maximize sensitivity while maintaining specificity.

What considerations are important when using TMEM222 antibodies for co-immunoprecipitation studies?

When designing co-immunoprecipitation (co-IP) experiments with TMEM222 antibodies, researchers should address these critical considerations:

• Membrane protein solubilization strategy:

  • Test multiple detergents (digitonin, CHAPS, DDM) at different concentrations

  • Optimize solubilization conditions to preserve protein-protein interactions

  • Consider crosslinking approaches (DSP, formaldehyde) to stabilize transient interactions

• Antibody selection and orientation:

  • Evaluate multiple TMEM222 antibodies for IP efficiency

  • Consider using antibodies targeting different epitopes to avoid interaction interface masking

  • Test both direct antibody coupling to beads and indirect capture with Protein A/G

• Validation controls:

  • Include IgG-matched negative control

  • Perform reverse IP when possible (IP suspected interaction partner, blot for TMEM222)

  • Include TMEM222-deficient sample as specificity control

• Buffer optimization:

  • Adjust salt concentration to minimize non-specific interactions while maintaining specific ones

  • Include appropriate protease and phosphatase inhibitors

  • Consider adding calcium chelators if studying calcium-dependent interactions

• Detection strategy:

  • For known interactions, use specific antibodies against interaction partners

  • For discovery, consider mass spectrometry analysis of co-IP products

  • Compare interactome of wild-type TMEM222 versus disease-associated variants

These methodological refinements will increase the likelihood of successfully capturing physiologically relevant TMEM222 protein complexes from neuronal cells or brain tissue.

How might combining TMEM222 antibodies with emerging technologies advance neurodevelopmental disorder research?

The integration of TMEM222 antibodies with cutting-edge technologies presents exciting opportunities for advancing our understanding of TMEM222-associated neurodevelopmental disorders:

• Spatial transcriptomics and proteomics:

  • Combine TMEM222 antibody staining with spatial transcriptomics to correlate protein localization with gene expression landscapes

  • Map TMEM222 distribution across brain regions at single-cell resolution

  • Identify cell populations particularly vulnerable to TMEM222 dysfunction

• Advanced imaging technologies:

  • Implement expansion microscopy with TMEM222 antibodies to visualize nanoscale organization within endosomes

  • Apply lattice light-sheet microscopy for dynamic tracking of TMEM222-positive structures in living neurons

  • Use correlative light and electron microscopy (CLEM) to place TMEM222 in ultrastructural context

• Organoid and assembloid models:

  • Develop brain organoids from patients with TMEM222 variants

  • Apply TMEM222 antibodies to track protein expression and localization during organoid development

  • Create assembloids connecting different brain regions to study TMEM222's role in circuit formation

• CRISPR-based approaches:

  • Generate knockin reporter lines tagging endogenous TMEM222

  • Create isogenic lines with patient-specific TMEM222 variants for direct comparison

  • Develop CRISPR activation/inhibition systems to modulate TMEM222 expression

• High-throughput drug screening:

  • Use TMEM222 antibodies as readouts in phenotypic screens

  • Identify compounds that restore normal TMEM222 localization or function in patient-derived cells

  • Develop assays that monitor endosomal trafficking as functional readouts

These integrated approaches will significantly accelerate our understanding of TMEM222 biology and potentially lead to therapeutic strategies for patients with TMEM222-associated neurodevelopmental disorders.

What are the most promising directions for therapeutic development targeting TMEM222 dysfunction?

Several promising therapeutic approaches could address TMEM222-associated neurodevelopmental disorders, based on current understanding of protein function and disease mechanisms:

• Gene therapy approaches:

  • AAV-mediated delivery of wild-type TMEM222 to affected brain regions

  • CRISPR-based correction of specific pathogenic variants

  • Antisense oligonucleotides to address splicing defects in intronic variants

• Small molecule modulators:

  • Compounds that stabilize mutant TMEM222 protein

  • Molecules targeting downstream endosomal trafficking pathways

  • Chaperone therapeutics to assist proper folding of variant proteins

• Endosomal pathway modulation:

  • Targeting compensatory endosomal trafficking pathways

  • Modulation of Rab GTPases that regulate early endosome dynamics

  • Compounds affecting endosomal maturation or recycling

• Cell-based therapies:

  • Neural stem cell transplantation to provide TMEM222 function

  • Exosome-based delivery of functional TMEM222 or compensatory factors

  • iPSC-derived neurons with corrected TMEM222 for potential transplantation

• Symptom-based approaches:

  • Target downstream signaling pathways affected by TMEM222 dysfunction

  • Address specific neurotransmitter imbalances that might result from trafficking defects

  • Behavioral and cognitive interventions tailored to specific deficits

TMEM222 antibodies will play a crucial role in these therapeutic development efforts by serving as tools to validate target engagement, assess restoration of normal protein localization, and monitor treatment effects on downstream pathways affected by TMEM222 dysfunction.