TMEM237 Antibody, HRP conjugated

What is TMEM237 and why is it significant in ciliopathy research?

TMEM237 (Transmembrane Protein 237, also known as ALS2CR4) is a tetraspan transmembrane protein that localizes to the ciliary transition zone (TZ) and plays a critical role in ciliogenesis and WNT signaling regulation. Its significance in research stems from its causal role in Joubert syndrome-14, a ciliopathy characterized by brain malformations and neurological symptoms .

The protein functions within a complex interaction network of transition zone proteins, including NPHP4, MKS-2/TMEM216, MKSR-1/B9D1, and MKSR-2/B9D2 . Loss of TMEM237 function results in defective ciliogenesis and dysregulated signaling pathways, particularly WNT signaling . The study of TMEM237 provides critical insights into transition zone biology and the molecular mechanisms underlying ciliopathies.

How does HRP conjugation affect antibody functionality compared to unconjugated TMEM237 antibodies?

HRP (Horseradish Peroxidase) conjugation to TMEM237 antibodies creates a direct detection system that offers several advantages over unconjugated antibodies:

What is the optimal protocol for ELISA using HRP-conjugated TMEM237 antibodies?

The optimal ELISA protocol for HRP-conjugated TMEM237 antibodies follows these methodological steps:

• Plate Coating: Coat 96-well plates with capture antigen or antibody (50-100 μL/well) in carbonate buffer (pH 9.6) overnight at 4°C.

• Blocking: Block with 3-5% BSA or non-fat milk in PBS-T (PBS + 0.05% Tween-20) for 1-2 hours at room temperature.

• Sample Addition: Add samples containing TMEM237 protein and incubate for 1-2 hours at room temperature.

• Primary Antibody Incubation: Apply HRP-conjugated TMEM237 antibody at the recommended dilution (typically 1:1000-1:20000) and incubate for 1-2 hours at room temperature.

• Washing: Wash 4-5 times with PBS-T to remove unbound antibody.

• Development: Add TMB substrate and incubate for 15-30 minutes.

• Stopping Reaction: Add stop solution (2N H₂SO₄).

• Detection: Read absorbance at 450 nm.

Critical parameters requiring optimization include antibody concentration, incubation times, and washing stringency. The HRP-conjugated format eliminates the need for a secondary antibody step, streamlining the protocol and potentially improving signal-to-noise ratios .

What sample preparation methods are most effective for detecting TMEM237 in Western blotting using HRP-conjugated antibodies?

Effective sample preparation for TMEM237 Western blotting requires particular attention to membrane protein extraction:

• Cell Lysis: Use buffer containing 1-2% non-ionic detergents (NP-40, Triton X-100) supplemented with protease inhibitor cocktail.

• Protein Denaturation: Add sample buffer containing SDS (2%) and reducing agent (β-mercaptoethanol).

• Heat Treatment: Heat at 70°C for 10 minutes (avoiding boiling, which can cause membrane protein aggregation).

• Loading Amount: Load 20-50 μg of total protein per lane.

• Molecular Weight Consideration: Though the calculated molecular weight of TMEM237 is 45.5 kDa , the observed molecular weight is approximately 72 kDa , likely due to post-translational modifications.

For Western blotting with HRP-conjugated TMEM237 antibodies, dilutions of 1:500-1:2000 are typically recommended . After transfer to a PVDF or nitrocellulose membrane, blocking with 5% non-fat milk or BSA in TBS-T for 1 hour at room temperature is critical before adding the HRP-conjugated antibody. Detection can be performed using enhanced chemiluminescence substrates optimized for HRP .

How can researchers validate the specificity of HRP-conjugated TMEM237 antibodies?

Comprehensive validation of HRP-conjugated TMEM237 antibodies should include multiple approaches:

Validation data should be thoroughly documented, as these controls are essential for publication quality data and ensuring experimental reproducibility.

What are common causes of background when using HRP-conjugated TMEM237 antibodies and how can they be mitigated?

Background issues when using HRP-conjugated TMEM237 antibodies can arise from multiple sources:

Implementing appropriate negative controls is crucial: (1) isotype controls, (2) secondary antibody-only controls, (3) blocking peptide competition controls, and (4) TMEM237-depleted samples. These controls help distinguish specific signal from background and validate experimental findings.

How can researchers optimize signal sensitivity for detecting low-abundance TMEM237 protein?

For detecting low-abundance TMEM237, several signal enhancement strategies can be employed:

• Tyramide Signal Amplification (TSA): This technique can increase sensitivity 10-100 fold by generating multiple tyramide radicals per HRP molecule.

• Enhanced Substrate Selection: For Western blotting, use femto-level ECL substrates with extended signal duration.

• Sample Enrichment Methods:

  • Immunoprecipitation prior to analysis

  • Cellular fractionation to isolate membrane fractions containing TMEM237

  • Concentration of samples using ultrafiltration

• Optimized Antibody Parameters:

  • Extended incubation (overnight at 4°C)

  • Reduced washing stringency while maintaining acceptable background

  • Optimization of blocking conditions

• Detection System Enhancement:

  • Use high-sensitivity digital imaging systems

  • Extend exposure times with low-noise detection

Each enhancement strategy should be systematically evaluated to determine the optimal combination for specific experimental conditions.

How can HRP-conjugated TMEM237 antibodies be utilized to investigate protein-protein interactions within the ciliary transition zone?

HRP-conjugated TMEM237 antibodies enable several sophisticated approaches for investigating transition zone protein interactions:

• Proximity-based Labeling: HRP-conjugated antibodies can generate biotin-phenol radicals that tag proteins in close proximity to TMEM237. These biotinylated proteins can then be isolated using streptavidin and identified via mass spectrometry.

• Co-immunoprecipitation with Direct Detection: Following immunoprecipitation, HRP-conjugated TMEM237 antibodies can directly detect interaction partners without requiring secondary antibodies, reducing background.

• In situ Proximity Ligation Assay (PLA): This technique can visualize and quantify protein-protein interactions with HRP signal amplification, providing spatial information about TMEM237 interactions.

• Electron Microscopy Localization: HRP's electron-dense reaction product allows precise ultrastructural localization of TMEM237 within the transition zone relative to other components.

These approaches are particularly valuable for investigating TMEM237's known interactions with transition zone proteins like RPGRIP1L/MKS5, NPHP4, MKS-2/TMEM216, MKSR-1/B9D1, and MKSR-2/B9D2 .

What methodological considerations are important when studying TMEM237 mutations associated with Joubert syndrome?

When studying TMEM237 mutations like c.890C>G (p.Ser297Ter) associated with Joubert syndrome, several methodological considerations are critical:

• Model System Selection:

  • Patient-derived cells

  • CRISPR-engineered cell lines harboring specific mutations

  • Animal models (zebrafish models as described in )

• Functional Assessments:

  • Ciliogenesis quantification (percentage of ciliated cells)

  • Cilia morphology (length, structure)

  • Transition zone integrity

  • WNT signaling activity (known to be dysregulated with TMEM237 loss )

• Localization Analysis:

  • Co-localization with other transition zone markers

  • Effect of mutations on TMEM237 localization

  • Dependency on RPGRIP1L/MKS5 for proper localization

• Rescue Experiments:

  • Complementation with wild-type TMEM237

  • Structure-function analysis with domain-specific constructs

• Interaction Analysis:

  • Effect of mutations on protein-protein interactions

  • Quantitative assessment of binding affinities

These approaches allow for comprehensive characterization of how specific TMEM237 mutations affect protein function and contribute to ciliopathy phenotypes.

How can researchers employ HRP-conjugated TMEM237 antibodies in multiplexed detection systems?

For multiplexed detection including TMEM237, researchers can implement these advanced techniques:

• Sequential TSA (Tyramide Signal Amplification):

  • Apply HRP-conjugated TMEM237 antibody

  • Develop with spectrally distinct fluorophore-conjugated tyramide

  • Inactivate HRP with hydrogen peroxide

  • Apply next antibody and repeat with different fluorophores

• Multi-enzyme Detection Systems:

  • Use HRP-conjugated TMEM237 antibodies alongside antibodies conjugated to different enzymes (alkaline phosphatase, glucose oxidase)

  • Develop with enzyme-specific substrates yielding distinct chromogenic or fluorescent products

• Antibody Stripping and Reprobing:

  • Detect TMEM237 with HRP-conjugated antibody

  • Document signal

  • Strip antibodies using appropriate buffer (e.g., glycine pH 2.5 or commercial stripping buffer)

  • Reprobe with additional antibodies

• Spectral Unmixing Approaches:

  • Use multiple fluorophores with overlapping spectra

  • Apply computational algorithms to separate signals

These approaches enable comprehensive mapping of protein networks within the ciliary transition zone, providing deeper insight into the molecular architecture disrupted in ciliopathies.

How should researchers interpret differences in TMEM237 detection patterns between antibodies targeting different epitopes?

When different TMEM237 antibodies yield varying detection patterns, consider these interpretations:

TMEM237 has known transcript variants , and antibodies targeting different regions (aa 59-78, 181-230, 251-300) may detect different forms. Additionally, post-translational modifications may mask certain epitopes while preserving others. Rather than viewing these differences as problematic, they can provide valuable insights into protein processing and modification in different contexts.

What controls and standards should be included when quantifying TMEM237 expression using HRP-conjugated antibodies?

Robust quantification of TMEM237 expression requires comprehensive controls:

• Quantification Standards:

  • Recombinant TMEM237 protein standards of known concentration

  • Standard curve covering the expected range of expression

  • Internal loading controls (housekeeping proteins for Western blot)

• Assay Validation Controls:

  • Negative controls: TMEM237 knockout/knockdown samples

  • Positive controls: Samples with verified TMEM237 expression

  • Technical replicates to assess reproducibility

• Normalization Strategy:

  • For Western blot: Total protein normalization or housekeeping proteins

  • For ELISA: Consistent total protein input and standard curve interpolation

  • For immunohistochemistry: Area-based or cell-based normalization

• Statistical Analysis:

  • Appropriate statistical tests based on data distribution

  • Multiple comparison corrections when analyzing multiple samples

  • Reporting of confidence intervals along with means

Implementing these controls ensures reliable quantification and facilitates comparison across experimental conditions and between different research groups.

How can HRP-conjugated TMEM237 antibodies contribute to understanding ciliopathy disease mechanisms?

HRP-conjugated TMEM237 antibodies enable several cutting-edge approaches for ciliopathy research:

• Patient-derived Organoid Models:

  • TMEM237 detection in disease-relevant 3D tissue structures

  • Correlation with ciliary function and tissue organization

  • Response to therapeutic candidates

• High-content Screening Applications:

  • Automated detection of TMEM237 localization changes

  • Screening for compounds that restore proper localization

  • Quantitative assessment of ciliary phenotypes

• In vivo Imaging:

  • Following conversion to compatible fluorescent formats

  • Real-time tracking of TMEM237 dynamics

  • Correlation with developmental processes disrupted in ciliopathies

• Systems Biology Integration:

  • Correlation of TMEM237 status with transcriptomic/proteomic profiles

  • Network analysis of transition zone components

  • Mathematical modeling of ciliary assembly

These approaches can significantly advance our understanding of how TMEM237 mutations lead to the complex phenotypes observed in Joubert syndrome and related ciliopathies .

What are the latest technical advances in antibody conjugation that may improve TMEM237-HRP antibody performance?

Recent technical advances with potential to improve TMEM237-HRP antibody performance include:

• Site-specific Conjugation:

  • Engineered antibodies with site-specific attachment points

  • Maintains native antigen-binding regions

  • More consistent conjugation ratios

• Alternative Enzyme Conjugates:

  • Evolved HRP variants with enhanced stability and activity

  • Smaller peroxidase enzymes that minimize steric hindrance

  • Dual-functionality enzymes enabling multiple detection modes

• Nanobody-based Approaches:

  • Single-domain antibody fragments conjugated to HRP

  • Smaller size improves tissue penetration

  • Reduced background in complex samples

• Controlled Orientation Conjugation:

  • Methods ensuring optimal orientation of antibody relative to HRP

  • Maximizes antigen binding while preserving enzyme activity

  • Improves detection sensitivity and consistency

• Stabilizing Technologies:

  • Enhanced formulations improving shelf-life

  • Freeze-dry compatible preparations

  • Temperature-stable variants for field applications

These technological advances promise to enhance sensitivity, specificity, and reproducibility in TMEM237 detection across diverse research applications.