TMEM231 Antibody

What is TMEM231 and why is it important in ciliopathy research?

TMEM231 is a two-pass transmembrane protein that functions as a critical component of the Meckel syndrome (MKS) complex at the ciliary transition zone, located between the basal body and axoneme. Its significance in ciliopathy research stems from its essential role in regulating the localization of ciliary membrane proteins . TMEM231 is evolutionarily conserved from Caenorhabditis elegans to mammals, indicating its fundamental importance in ciliary biology . Mutations in TMEM231 have been identified in patients with orofaciodigital syndrome type 3 (OFD3) and Meckel syndrome, making it a crucial protein for understanding the molecular basis of these developmental disorders . Researchers investigating ciliopathies typically employ TMEM231 antibodies to examine its localization patterns, protein interactions, and functional consequences of its mutation or depletion.

What are the validated applications for TMEM231 antibodies in research?

Based on current validation data, TMEM231 antibodies have been successfully employed in multiple experimental applications. Specifically, the rabbit polyclonal TMEM231 antibody (23731-1-AP) has been validated for immunohistochemistry (IHC), immunofluorescence (IF)/immunocytochemistry (ICC), and enzyme-linked immunosorbent assay (ELISA) . For IHC applications, this antibody has been successfully tested on human pancreatic tissue with recommended antigen retrieval using TE buffer (pH 9.0) or alternatively with citrate buffer (pH 6.0) . For IF/ICC applications, positive detection has been confirmed in MDCK cells . Researchers should note that reactivity has been specifically demonstrated with human and canine samples, which should inform experimental design when working with different model organisms .

What is the molecular profile of TMEM231, and how does this impact antibody selection?

TMEM231 (also known as hypothetical protein FLJ22167) consists of 345 amino acids with a calculated molecular weight of 39 kDa . The protein is encoded by the gene with ID 79583 (NCBI) and has a GenBank Accession Number of BC010609 . When selecting antibodies for TMEM231 detection, researchers should consider these molecular characteristics to ensure proper target recognition. The antibody's immunogen sequence (for example, the TMEM231 fusion protein Ag20669 used to generate the Proteintech antibody) should be evaluated for species cross-reactivity and epitope accessibility in different experimental conditions . Understanding TMEM231's transmembrane topology is also crucial when choosing antibodies, as epitopes may be differentially accessible depending on protein conformation and membrane integration.

How can researchers distinguish between different ciliary transition zone complexes when using TMEM231 antibodies?

Distinguishing between different ciliary transition zone complexes requires sophisticated co-localization studies. TMEM231 functions within the MKS complex alongside other proteins including B9d1, Mks1, Tctn1, Tctn2, Tctn3, Cc2d2a (Mks6), and Tmem17 . To differentiate between transition zone complexes, researchers should:

• Perform multi-color immunofluorescence using antibodies against TMEM231 alongside markers for other transition zone complexes (e.g., NPHP complex proteins).

• Analyze co-immunoprecipitation data quantitatively to determine the stoichiometry of TMEM231 with other MKS complex components.

• Implement super-resolution microscopy techniques (STED, STORM, or PALM) to precisely map TMEM231 localization relative to other transition zone proteins.

• Use proximity ligation assays to verify protein-protein interactions between TMEM231 and other transition zone components in situ.

This approach allows researchers to distinguish the MKS complex from other transition zone assemblies and determine how TMEM231 contributes to the architectural organization of this ciliary region.

What considerations should be made when designing experiments to study TMEM231 mutations using antibodies?

When studying TMEM231 mutations using antibodies, researchers should consider several critical factors:

• Epitope accessibility: Disease-associated mutations may affect protein folding or complex formation. Researchers should evaluate whether mutations might alter epitope accessibility for the selected antibody.

• Mutation-specific effects: Different mutations have distinct molecular consequences. For instance, the p.Asn90Ile and p.Pro125Ala mutations were shown to compromise TMEM231 protein levels, while p.Leu81Phe and p.Ala216Pro did not significantly affect protein stability but still disrupted function .

• Interaction analysis: All four disease-associated TMEM231 mutant proteins retained their ability to interact with B9d1, despite compromising transition zone organization . Researchers should design co-immunoprecipitation experiments to assess how mutations affect specific protein interactions.

• Localization studies: Mutation-specific localization defects require carefully designed immunofluorescence experiments. For example, the p.Asn90Ile mutation causes mislocalization of B9d1 away from the transition zone to the centrosome, suggesting potential dominant-negative effects .

• Functional readouts: Antibody-based assays should be coupled with functional analyses measuring ciliary membrane composition, such as Arl13b localization, which is disrupted in TMEM231-deficient cells .

These considerations will help researchers accurately characterize the molecular consequences of TMEM231 mutations and their relationship to disease phenotypes.

What are the optimal conditions for immunohistochemical detection of TMEM231 in different tissue types?

For optimal immunohistochemical detection of TMEM231 across different tissue types, researchers should follow these methodological guidelines:

Recommended IHC Protocol:

• Fixation: Use 4% paraformaldehyde for tissue fixation to preserve epitope accessibility while maintaining tissue architecture.

• Antigen retrieval: Primary recommendation is TE buffer at pH 9.0, with citrate buffer at pH 6.0 as an alternative . Optimization for specific tissue types may be necessary.

• Antibody dilution: For IHC applications, use dilutions ranging from 1:20 to 1:200 . Titration is recommended for each specific tissue type.

• Detection system: Use a polymer-based detection system for enhanced sensitivity with minimal background.

• Controls: Include appropriate positive controls (human pancreas has been validated) and negative controls (primary antibody omission and isotype controls).

• Tissue-specific considerations:

  • For kidney tissue: Special attention to corticomedullary junction where TMEM231-related cysts develop in disease models

  • For brain tissue: Extended fixation times may require adjusted antigen retrieval parameters

  • For developing tissues: Stage-specific expression patterns may require adjusted antibody concentrations

Researchers should note that TMEM231 detection may vary between ciliated and non-ciliated cell types within the same tissue, requiring careful analysis and interpretation.

How can researchers effectively use TMEM231 antibodies in co-immunoprecipitation experiments to study protein interactions?

For effective co-immunoprecipitation experiments using TMEM231 antibodies, researchers should implement the following methodological approach:

Recommended Co-IP Protocol:

• Cell lysis conditions: Use a buffer containing 50 mM Hepes (pH 7.4), 300 mM KCl, 1 mM EGTA, 1 mM MgCl₂, 10% glycerol, 0.3% NP-40, 0.5 mM DTT, and protease and phosphatase inhibitors .

• Clearing procedure: Clear lysates by centrifugation at 3,500 g for 20 minutes to remove cell debris .

• Incubation parameters: Incubate cleared lysates with antibody-conjugated beads (e.g., FLAG-M2 beads for tagged proteins) for 4-6 hours at 4°C with gentle rotation .

• Washing protocol: Perform three washes with lysis buffer to reduce non-specific binding .

• Elution method: Elute bound proteins by resuspending beads in 6× SDS-PAGE loading buffer followed by denaturation at 100°C for 5 minutes .

• Gel resolution: Resolve immunoprecipitations on 4-15% TGX gradient gels for optimal separation of TMEM231 and interacting partners .

• Detection strategy: Transfer to PVDF membrane and probe with appropriate antibodies, including those against known MKS complex components like B9d1, Mks1, Tctn1, and Cc2d2a.

• Validation approach: Perform reciprocal co-IPs to confirm interactions, and consider including negative controls (unrelated proteins) to validate specificity.

This protocol has been successfully employed to demonstrate interactions between TMEM231 and multiple components of the MKS complex .

What are the technical challenges and solutions for immunofluorescence detection of TMEM231 at the ciliary transition zone?

Technical Challenges and Solutions for TMEM231 Immunofluorescence:

• Challenge: Small size of the transition zone leading to difficult visualization
  Solution: Implement super-resolution microscopy techniques or structured illumination microscopy to resolve the narrow transition zone region (approximately 0.2-0.5 μm).

• Challenge: Co-detection with other transition zone markers
  Solution: Use spectrally distinct fluorophores and sequential antibody incubations to prevent steric hindrance between antibodies targeting proximal epitopes.

• Challenge: Signal-to-noise ratio in multiciliated tissues
  Solution: For IF/ICC applications, use dilutions ranging from 1:200 to 1:800 combined with confocal microscopy to improve signal clarity.

• Challenge: Epitope masking within protein complexes
  Solution: Test multiple fixation and permeabilization protocols; mild detergents (0.1% Triton X-100 or 0.1% saponin) may preserve complex architecture while allowing antibody access.

• Challenge: Distinguishing TMEM231 from other transition zone proteins
  Solution: Use validated ciliary markers, including:

  • Acetylated tubulin for the axoneme

  • Gamma-tubulin for the basal body

  • Specific markers for the transition fiber (Cep164) and Y-links

• Challenge: Cell type-specific variations in ciliary architecture
  Solution: Optimize protocols for each cell type; MDCK cells have been validated for TMEM231 antibody (23731-1-AP) in IF/ICC applications .

These technical approaches enable reliable visualization of TMEM231 at the transition zone, facilitating accurate analysis of its role in ciliary biology and disease processes.

How should researchers interpret variations in TMEM231 antibody staining patterns across different ciliated tissues?

Interpretation of TMEM231 staining patterns across different ciliated tissues requires careful consideration of several biological and technical factors:

• Cell type-specific ciliary architecture:

  • Primary cilia in renal epithelial cells may show different TMEM231 distribution patterns compared to motile cilia in respiratory epithelium

  • Brain ventricle ependymal cells may exhibit distinct TMEM231 localization patterns compared to choroid plexus cells

• Developmental timing effects:

  • TMEM231 expression and localization may vary during ciliogenesis stages

  • Embryonic versus adult tissues may show differential staining intensities reflecting developmental regulation

• Disease-specific alterations:

  • In ciliopathy contexts, researchers should differentiate between primary defects in TMEM231 localization and secondary consequences of disrupted ciliary architecture

  • Quantitative image analysis should be employed to detect subtle changes in TMEM231 distribution

• Technical interpretation guidelines:

  • Expected staining pattern: Discrete punctate localization at the ciliary base, specifically at the transition zone between the basal body and axoneme

  • Abnormal patterns: Diffuse cytoplasmic staining, mislocalization to basal bodies, or complete absence from cilia

  • Control comparisons: Always analyze TMEM231 staining in relation to established transition zone markers

• Quantitative assessment approaches:

  • Measure co-localization coefficients with other transition zone proteins (Mks1, B9d1)

  • Analyze the ratio of transition zone to cytoplasmic signal intensity

  • Evaluate the percentage of cilia with detectable TMEM231 staining

These interpretation guidelines will help researchers accurately assess TMEM231 localization across different experimental and pathological contexts.

What approaches should be used to analyze contradictory results from TMEM231 antibody experiments?

When faced with contradictory results from TMEM231 antibody experiments, researchers should implement a systematic troubleshooting and analytical approach:

• Antibody validation verification:

  • Confirm antibody specificity using TMEM231 knockout or knockdown controls

  • Test multiple antibodies targeting different TMEM231 epitopes

  • Verify results using tagged TMEM231 constructs in parallel with antibody detection

• Technical variables assessment:

  • Systematically evaluate fixation conditions, permeabilization methods, and antigen retrieval parameters

  • Compare detection methods (direct immunofluorescence versus amplification systems)

  • Ensure appropriate dilution ranges are tested (1:20-1:200 for IHC; 1:200-1:800 for IF/ICC)

• Biological variables consideration:

  • Assess cell cycle stage effects on ciliary formation and TMEM231 localization

  • Evaluate cell confluency and serum starvation conditions that affect ciliogenesis

  • Consider species-specific differences (antibody 23731-1-AP is validated for human and canine samples)

• Data integration strategies:

  • Implement multiple complementary techniques (IF, IHC, western blot, mass spectrometry)

  • Perform functional assays to correlate TMEM231 localization with ciliary membrane composition

  • Use genetic approaches (rescue experiments with wild-type versus mutant TMEM231) to validate antibody findings

• Statistical analysis approach:

  • Quantify results across multiple experiments and biological replicates

  • Apply appropriate statistical tests to determine significance of observed differences

  • Consider power analysis to ensure adequate sample sizes

How can TMEM231 antibodies be utilized to study the functional impact of disease-associated mutations?

TMEM231 antibodies can be employed in sophisticated experimental paradigms to characterize the functional consequences of disease-associated mutations:

Recommended Experimental Approach:

• Rescue assay design:

  • Generate TMEM231-deficient cell lines using CRISPR/Cas9

  • Express wild-type or mutant TMEM231 constructs (p.Leu81Phe, p.Asn90Ile, p.Pro125Ala, or p.Ala216P)

  • Assess protein localization using immunofluorescence with antibodies against TMEM231 and other transition zone markers

  • Quantify rescue efficiency based on restoration of ciliary membrane composition (e.g., Arl13b localization)

• Protein stability analysis:

  • Employ cycloheximide chase assays with TMEM231 antibody detection to determine if mutations affect protein half-life

  • Compare steady-state levels of wild-type versus mutant proteins using western blot analysis

  • Correlate stability defects with observed hypomorphic phenotypes in patient-derived cells

• Complex assembly assessment:

  • Use antibodies against TMEM231 and other MKS complex components in co-immunoprecipitation experiments

  • Quantify the efficiency with which mutant forms of TMEM231 interact with partners like B9d1

  • Analyze transition zone recruitment of MKS complex components in cells expressing mutant TMEM231

• Functional readout development:

  • Establish quantitative assays for ciliary transition zone barrier function

  • Measure cilia-dependent signaling pathways (Hedgehog, PDGF, etc.) in cells expressing disease-associated TMEM231 variants

  • Correlate molecular defects with cellular and organismal phenotypes

These approaches have revealed that disease-associated TMEM231 mutations are often hypomorphic, retaining some functionality while compromising critical aspects of transition zone organization and ciliary membrane composition .

What methodological approaches can be used to study TMEM231 protein dynamics at the ciliary transition zone?

To investigate TMEM231 protein dynamics at the ciliary transition zone, researchers should consider these advanced methodological approaches:

• Live imaging techniques:

  • Generate fluorescently-tagged TMEM231 constructs that maintain native localization and function

  • Implement photobleaching approaches (FRAP, FLIP) to measure protein turnover rates at the transition zone

  • Use photoactivatable or photoconvertible fluorophores to track newly synthesized TMEM231 recruitment to cilia

• Super-resolution microscopy applications:

  • Apply STORM, PALM, or STED microscopy to visualize TMEM231 nanoscale organization

  • Implement multi-color super-resolution to map TMEM231 position relative to other transition zone proteins

  • Correlate super-resolution data with electron microscopy findings to establish structure-function relationships

• Proximity labeling strategies:

  • Fuse TMEM231 to BioID, TurboID, or APEX2 to identify proximal proteins in living cells

  • Compare proximity interactomes between wild-type and disease-associated TMEM231 variants

  • Validate novel interactions using conventional antibody-based approaches

• Transition zone barrier function assays:

  • Develop quantitative measures of membrane protein diffusion across the transition zone

  • Assess rates of entry and exit for ciliary membrane proteins in cells with wild-type versus mutant TMEM231

  • Correlate barrier function with transition zone ultrastructure

• Optogenetic perturbation approaches:

  • Create light-sensitive TMEM231 fusion proteins to acutely disrupt transition zone organization

  • Measure immediate consequences on ciliary protein composition and signaling

  • Compare acute versus chronic loss of TMEM231 function

These methodological approaches provide powerful tools for dissecting the dynamic role of TMEM231 in maintaining the ciliary transition zone and regulating ciliary composition.

What are the validated dilution ranges and application conditions for TMEM231 antibodies?

The following table summarizes the recommended dilution ranges and application conditions for the rabbit polyclonal TMEM231 antibody (23731-1-AP):

Researchers should note that these parameters should be considered starting points, and optimal conditions may vary based on specific experimental contexts . It is recommended that the antibody be titrated in each testing system to obtain optimal results .

How should researchers troubleshoot weak or non-specific signals when using TMEM231 antibodies?

When encountering weak or non-specific signals with TMEM231 antibodies, researchers should implement the following troubleshooting approaches:

For Weak Signal:

• Antibody concentration adjustment:

  • Increase primary antibody concentration within the recommended range (1:20-1:200 for IHC; 1:200-1:800 for IF/ICC)

  • Extend primary antibody incubation time (overnight at 4°C versus 1-2 hours at room temperature)

• Antigen retrieval optimization:

  • Compare heat-induced epitope retrieval using TE buffer (pH 9.0) versus citrate buffer (pH 6.0)

  • Test different retrieval times and temperatures

• Detection system enhancement:

  • Implement signal amplification methods (tyramide signal amplification, polymer-based detection)

  • Use brighter fluorophores or higher sensitivity enzyme substrates

• Sample preparation refinement:

  • Optimize fixation conditions to preserve epitope accessibility

  • Adjust permeabilization parameters to facilitate antibody penetration

For Non-specific Signal:

• Blocking protocol enhancement:

  • Test different blocking agents (BSA, normal serum, commercial blocking solutions)

  • Extend blocking time to reduce background

• Antibody specificity verification:

  • Include appropriate negative controls (TMEM231 knockout/knockdown samples)

  • Perform pre-absorption controls with immunizing peptide

• Washing optimization:

  • Increase wash duration and number of washing steps

  • Add detergents at appropriate concentrations to reduce non-specific binding

• Dilution optimization:

  • Test serial dilutions to identify optimal signal-to-noise ratio

  • Consider using fragment antibodies (Fab) if steric issues are suspected

These troubleshooting approaches should be systematically implemented and documented to establish optimal conditions for TMEM231 detection in specific experimental contexts.