TMEM237 Antibody, FITC conjugated

What is TMEM237 and why is it studied in research?

TMEM237 (Transmembrane protein 237), also known as ALS2CR4 (Amyotrophic lateral sclerosis 2 chromosomal region candidate gene 4 protein), is a tetraspanin protein that localizes to the ciliary transition zone (TZ) . It plays a critical role in ciliogenesis and is involved in WNT signaling pathways . The protein has gained significant research interest because mutations in TMEM237 cause Joubert syndrome-14, a ciliopathy characterized by brain malformations and various systemic manifestations .

TMEM237 has been studied in various model systems including mammalian cells, zebrafish (Danio rerio), and nematodes (Caenorhabditis elegans), revealing its evolutionarily conserved function in the ciliary transition zone . Research indicates that TMEM237 interacts with other transition zone proteins including NPHP4, MKS2, MKSR1/B9D1, MKSR2/B9D2, and MKS5/RPGRIP1L to control basal body-transition zone anchoring to the membrane and ciliogenesis .

What applications is TMEM237 Antibody, FITC conjugated best suited for?

TMEM237 Antibody, FITC conjugated is primarily designed for applications requiring fluorescent detection, including:

The FITC conjugation eliminates the need for secondary antibody incubation, reducing experimental time and potential cross-reactivity issues . This makes it particularly valuable for multicolor staining protocols where antibody species limitations exist .

What is the recommended storage protocol for maintaining antibody activity?

To maintain optimal activity of TMEM237 Antibody, FITC conjugated:

• Store at -20°C or -80°C upon receipt .

• Avoid repeated freeze-thaw cycles which can reduce antibody activity and increase background .

• For short-term storage (1-2 weeks), the antibody can be kept at 4°C protected from light.

• The antibody is typically supplied in a buffer containing 50% glycerol, 0.01M PBS, pH 7.4, and 0.03% Proclin 300 as preservative .

FITC is sensitive to photobleaching, so minimize exposure to light during storage and handling to preserve fluorescence intensity .

What is the optimal protocol for immunofluorescence staining using TMEM237 Antibody, FITC conjugated?

Detailed Immunofluorescence Protocol:

• Cell preparation:

  • Seed cells at 2 × 10⁴ cells/well on glass coverslips in six-well plates .

  • Allow cells to reach desired confluence (typically 70-80%).

• Fixation options:

  • Ice-cold methanol (5 min at -20°C) - preferred for ciliary structures

  • 2% paraformaldehyde (20 min at room temperature)

• Permeabilization:

  • If using paraformaldehyde fixation, permeabilize with 0.1% Triton X-100 in PBS for 5-10 minutes.

  • Methanol-fixed cells do not require additional permeabilization.

• Blocking:

  • Block with 5% normal serum (from the same species as the secondary antibody) in PBS for 30-60 minutes.

• TMEM237 Antibody, FITC conjugated incubation:

  • Dilute antibody (typically 1:200-1:1000) in blocking buffer .

  • Incubate overnight at 4°C or 1-2 hours at room temperature in a humid chamber.

• Nuclear counterstaining:

  • DAPI (4′,6-diamidino-2-phenylindole) can be used to visualize nuclei .

• Mounting and imaging:

  • Mount slides using anti-fade mounting medium.

  • Image using appropriate fluorescence filters (FITC: excitation ~495 nm, emission ~520 nm) .

Note: For co-localization studies with ciliary markers, consider using antibodies against polyglutamylated tubulin (GT-335) as a ciliary shaft marker .

How can researchers optimize signal-to-noise ratio when using TMEM237 Antibody, FITC conjugated?

To achieve optimal signal-to-noise ratio:

• Antibody titration:

  • Perform a titration experiment (1:50 to 1:1000) to determine optimal concentration.

  • The ideal concentration provides maximum specific signal with minimal background.

• Fixation optimization:

  • Compare methanol vs. paraformaldehyde fixation for your specific cell type.

  • TMEM237 localization to the ciliary transition zone may be better preserved with certain fixation methods .

• Background reduction strategies:

  • Include 0.1-0.3% Triton X-100 in antibody diluent to reduce non-specific membrane binding.

  • If high background persists, pre-adsorb antibody with fixed/permeabilized cells lacking the target.

  • Include additional washing steps (minimum 3× 5 minutes with PBS-T).

• Control for autofluorescence:

  • Include an unstained sample to assess cellular autofluorescence.

  • Consider treatment with sodium borohydride (NaBH₄) to reduce autofluorescence from aldehyde fixatives.

• Photobleaching prevention:

  • Minimize exposure to light during all steps.

  • Use anti-fade mounting media containing agents like p-phenylenediamine or proprietary anti-fade compounds.

What controls should be included when using TMEM237 Antibody, FITC conjugated?

Essential controls for TMEM237 Antibody, FITC conjugated experiments:

For RNAi knockdown validation, researchers can use previously validated siRNA sequences: duplex 1 5′-GGAUCUUAGUGAAGAGUUATT and duplex 2 5′-GAACGAAAACGGCAUUGAUTT .

How can TMEM237 Antibody, FITC conjugated be used to study ciliopathies like Joubert syndrome?

TMEM237 Antibody, FITC conjugated provides a valuable tool for investigating ciliopathies through multiple methodological approaches:

• Patient-derived cell studies:

  • Compare TMEM237 localization in fibroblasts from patients with Joubert syndrome-14 versus control fibroblasts .

  • Examine co-localization with other transition zone proteins using confocal microscopy.

  • Investigate ciliogenesis defects by counting percentage of ciliated cells and measuring ciliary length.

• Signaling pathway analysis:

  • Investigate WNT signaling dysregulation using TMEM237 Antibody, FITC conjugated in conjunction with reporters like Topflash luciferase assay .

  • Examine potential crosstalk between TMEM237 and other ciliopathy-associated proteins.

• Rescue experiments:

  • Perform transfection with wild-type TMEM237 in patient cells and analyze restoration of protein localization and function.

  • Quantify rescue effects on ciliogenesis and related phenotypes.

• Animal model studies:

  • Use the antibody to validate TMEM237 knockdown/knockout in zebrafish or mouse models .

  • Correlate phenotypes with cellular and molecular defects.

Research has demonstrated that TMEM237-deficient cells have defects in ciliogenesis and show deregulation of Wnt signaling pathways , providing important mechanistic insights into Joubert syndrome pathogenesis.

What are the technical considerations for using TMEM237 Antibody, FITC conjugated in flow cytometry?

When using TMEM237 Antibody, FITC conjugated for flow cytometry:

• Sample preparation:

  • For intracellular staining of TMEM237, cells must be fixed and permeabilized (methanol or commercial permeabilization kits).

  • Gentle fixation conditions may better preserve epitope recognition.

• FITC properties and considerations:

  • FITC has excitation maximum at 495 nm and emission at 520 nm, compatible with standard 488 nm lasers .

  • FITC is pH-sensitive (optimal at pH > 7.0) and susceptible to photobleaching.

  • Typical conjugation results in 3-6 FITC molecules per antibody; higher conjugation can cause solubility problems and quenching .

• Panel design:

  • FITC has considerable spectral overlap with PE; appropriate compensation is essential.

  • FITC works well in combination with APC, PE-Cy5, and PE-Cy7.

• Titration and controls:

  • Perform antibody titration to determine optimal concentration.

  • Use isotype control (rabbit IgG-FITC) at the same concentration.

  • Include unstained cells to establish autofluorescence baseline.

• Data analysis:

  • For ciliary proteins like TMEM237, expect potentially low signal due to limited ciliary expression.

  • Consider using ciliated cell enrichment methods before analysis.

How does TMEM237 interact with other ciliary transition zone proteins and what methodologies can reveal these interactions?

TMEM237 has been shown to interact with several other transition zone proteins. Research methodologies to study these interactions include:

• Co-immunoprecipitation:

  • TMEM237 requires RPGRIP1L/MKS5 for proper transition zone localization .

  • IP-Western blot analysis can reveal direct protein-protein interactions.

• Confocal microscopy co-localization:

  • Use TMEM237 Antibody, FITC conjugated with other antibodies against transition zone proteins.

  • High-resolution imaging techniques such as structured illumination microscopy (SIM) or STORM can provide detailed spatial relationships.

• Genetic interaction studies:

  • In C. elegans, jbts-14 (TMEM237 homolog) genetically interacts with nphp-4 to control basal body-transition zone anchoring .

  • Similar approaches can be applied in mammalian cell culture using combinatorial siRNA knockdowns.

• Functional rescue experiments:

  • Expression of wild-type protein can rescue phenotypes in knockdown/knockout models.

  • These experiments help establish hierarchy of protein interactions.

Known interaction partners include:

• NPHP4 (genetic interaction demonstrated in C. elegans)

• MKS5/RPGRIP1L (required for TMEM237 localization)

• MKS-2/TMEM216

• MKSR-1/B9D1

• MKSR-2/B9D2

What are common issues encountered with TMEM237 Antibody, FITC conjugated and how can they be resolved?

For particularly challenging applications, consider:

• Using signal amplification systems

• Comparing results with non-conjugated primary antibody plus secondary detection

• Validating with alternative detection methods (e.g., Western blot)

How can researchers validate the specificity of TMEM237 Antibody, FITC conjugated?

Comprehensive validation of TMEM237 Antibody, FITC conjugated should include:

• Genetic approaches:

  • siRNA knockdown: Use validated siRNA sequences (duplex 1: 5′-GGAUCUUAGUGAAGAGUUATT and duplex 2: 5′-GAACGAAAACGGCAUUGAUTT) to reduce TMEM237 expression and confirm corresponding reduction in antibody signal.

  • CRISPR/Cas9 knockout: Generate complete knockout cells for definitive negative control.

• Immunoblot analysis:

  • Confirm single band of expected molecular weight (~45 kDa for human TMEM237).

  • Compare pattern with different antibody targeting different epitope of same protein.

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide before staining to block specific binding.

  • Signal should be substantially reduced or eliminated.

• Subcellular localization:

  • Confirm expected localization to ciliary transition zone .

  • Co-staining with established ciliary markers (e.g., acetylated tubulin, polyglutamylated tubulin) .

• Cross-species validation:

  • Test reactivity in multiple species if antibody is predicted to cross-react.

  • TMEM237 is evolutionarily conserved across species including human, mouse, zebrafish, and C. elegans .

How can TMEM237 Antibody, FITC conjugated be used in multi-color immunofluorescence experiments?

Multi-color immunofluorescence strategy with TMEM237 Antibody, FITC conjugated:

• Compatible fluorophore combinations:

  Fluorophore	Excitation (nm)	Emission (nm)	Compatible with FITC
  FITC (TMEM237)	495	520	-
  Alexa Fluor 568	578	603	Yes
  Alexa Fluor 647	650	665	Yes
  DAPI	358	461	Yes

• Suggested marker combinations for ciliary studies:

  • TMEM237 Antibody, FITC conjugated (transition zone)

  • Acetylated α-tubulin (Alexa Fluor 568) - ciliary axoneme

  • γ-tubulin (Alexa Fluor 647) - basal body

  • DAPI - nucleus

• Sequential staining protocol:

  • Apply TMEM237 Antibody, FITC conjugated first

  • Add additional primary antibodies (non-rabbit origin to avoid cross-reactivity)

  • Use appropriate species-specific secondary antibodies

  • Add nuclear counterstain last

• Image acquisition considerations:

  • Capture single-color controls for spectral unmixing

  • Image channels sequentially rather than simultaneously to minimize bleed-through

  • Begin with longest wavelength (least photobleaching) and end with FITC channel

• Analysis approaches:

  • Line scan analysis across cilia to determine precise localization of TMEM237 relative to other markers

  • 3D reconstruction to visualize spatial relationships between transition zone proteins

What are the most promising future research directions for TMEM237 studies?

Recent findings about TMEM237 suggest several promising research directions:

• Therapeutic development for ciliopathies:

  • Using TMEM237 Antibody, FITC conjugated to screen small molecule libraries for compounds that rescue localization defects

  • Investigating gene therapy approaches to restore TMEM237 function

• Wnt signaling pathway interactions:

  • TMEM237 has been implicated in WNT signaling regulation

  • Further research into how TMEM237 modulates canonical and non-canonical Wnt pathways

  • Exploration of potential therapeutic targeting of these pathways in ciliopathies

• Transition zone assembly mechanisms:

  • Deeper investigation of the hierarchical assembly of transition zone components

  • Time-lapse imaging of fluorescently-tagged TMEM237 during ciliogenesis

  • Structural biology approaches to understand transition zone architecture

• Broader ciliopathy connections:

  • Investigation of TMEM237 in other ciliopathies beyond Joubert syndrome

  • Exploration of potential roles in cancer and developmental disorders

  • Population studies to identify additional pathogenic variants

• Systems biology integration:

  • Proteomics and interactome studies to comprehensively map TMEM237 interactions

  • Computational modeling of transition zone dynamics

  • Integration of multi-omics data to understand TMEM237's role in cellular homeostasis

How can researchers use TMEM237 Antibody, FITC conjugated in super-resolution microscopy?

The transition zone where TMEM237 localizes is approximately 200-400 nm in length, making super-resolution microscopy ideal for detailed structural studies:

• Sample preparation optimization:

  • Use high-precision (No. 1.5H, 170 ± 5 μm) coverslips

  • Consider rhodamine fiducials for drift correction

  • Optimize fixation to preserve ultrastructure (glutaraldehyde may be added at low concentrations)

• Technique-specific considerations:

  Super-resolution Technique	Key Advantages for TMEM237 Studies	Special Considerations
  STED (Stimulated Emission Depletion)	Live-cell compatible, direct imaging	FITC is not ideal; consider using non-conjugated primary with Alexa Fluor 488 secondary
  STORM/dSTORM	Highest resolution (~20 nm), good for precise localization	Requires special buffers, FITC not optimal (photobleaching)
  SIM (Structured Illumination)	Compatible with standard sample prep, multiple colors	Lower resolution than other techniques but FITC-compatible
  Expansion Microscopy	Physical expansion of sample, works with standard microscopes	Compatible with FITC, protocol may need optimization

• Analysis approaches:

  • Quantify radial distribution of TMEM237 around transition zone

  • Measure co-localization with other TZ proteins at nanometer scale

  • Determine precise stoichiometry of protein complexes

• Recommended protein partners for co-localization:

  • RPGRIP1L/MKS5 (required for TMEM237 localization)

  • NPHP4 (genetic interaction partner)

  • MKS-2/TMEM216, MKSR-1/B9D1, MKSR-2/B9D2 (functional interaction partners)

What methodologies can be used to study TMEM237's role in WNT signaling pathways?

TMEM237 has been implicated in regulating WNT signaling pathways . The following methodologies can be used to investigate this function:

• Reporter assays:

  • Topflash luciferase assay to measure canonical Wnt activity

  • Protocol: Transfect cells with Topflash firefly luciferase construct (or Fopflash as negative control), TMEM237 expression construct, and Renilla luciferase internal control

  • Stimulate with Wnt3A-conditioned media to activate canonical pathway or Wnt5A for non-canonical pathway

  • Compare wild-type vs. TMEM237-depleted cells

• RhoA activation assays:

  • Use pull-down assays with GST fusion protein of Rho effector rhotekin

  • Western blot for total RhoA and activated GTP-bound isoform

  • This approach reveals TMEM237's impact on non-canonical Wnt/PCP pathway

• Immunofluorescence for pathway components:

  • Use TMEM237 Antibody, FITC conjugated with antibodies against:

    • β-catenin (nuclear translocation indicates canonical pathway activation)

    • Dishevelled (membrane recruitment indicates pathway activation)

    • RhoA (for non-canonical pathway)

• Live cell imaging:

  • Monitor dynamics of fluorescently-tagged Wnt pathway components in TMEM237 knockdown cells

  • Measure response times and magnitudes after Wnt stimulation

• Ciliary localization of Wnt receptors:

  • Investigate whether TMEM237 affects localization of Wnt receptors (Frizzled, LRP5/6) to primary cilia

  • Quantify receptor enrichment at cilia in control vs. TMEM237-depleted cells