TMEM132A Antibody

What is TMEM132A and what are its known biological functions?

TMEM132A (Transmembrane Protein 132A) is a transmembrane protein with a calculated molecular weight of approximately 110 kDa (1023 amino acids) . Recent research has established TMEM132A as a multifunctional protein with several critical biological roles:

• Regulator of Wnt signaling pathway through stabilization of Wnt ligands and enhancement of WLS-Wnt ligand interactions

• Crucial factor in neural tube development, with knockout mice exhibiting neural tube defects

• Mediator of cell migration through regulation of integrin pathways and cytoskeletal remodeling

• Stabilizer of the Wnt co-receptor LRP6, affecting canonical Wnt/β-catenin signaling

The protein predominantly localizes to the endoplasmic reticulum and plasma membrane, particularly at pseudopodia and cell-cell junction regions .

What applications are TMEM132A antibodies commonly used for?

TMEM132A antibodies have been validated for multiple research applications, with varying dilution requirements:

When designing experiments, it is advisable to titrate antibodies to obtain optimal signal-to-noise ratios for your specific sample type .

What species reactivity do TMEM132A antibodies typically exhibit?

Available TMEM132A antibodies demonstrate cross-reactivity with several species:

When studying TMEM132A in non-human models, it is essential to verify species cross-reactivity and validate antibody performance in your specific experimental system .

What is the expected molecular weight band for TMEM132A in Western blotting?

• Post-translational modifications

• Tissue-specific isoforms

• Species differences

Positive controls for Western blot validation include:

• HaCaT cells

• MCF-7 cells

• SH-SY5Y cells

• U-87 MG cells

• Fetal human brain tissue

• Mouse and rat brain tissue

How should I optimize Western blot conditions for TMEM132A detection?

For optimal TMEM132A detection by Western blot:

• Sample preparation:

  • Use brain tissue (human, mouse, or rat) or neural cell lines (SH-SY5Y, U-87 MG) as positive controls

  • Include protease inhibitors in lysis buffer to prevent degradation

• Gel electrophoresis:

  • Use 8-10% SDS-PAGE gels to properly resolve the 100-140 kDa TMEM132A protein

• Transfer conditions:

  • Optimize transfer time for high molecular weight proteins (typically longer transfer times or lower current)

• Antibody incubation:

  • Starting dilution: 1:2000-1:10000

  • Primary antibody incubation: Overnight at 4°C

  • Secondary antibody: Anti-rabbit IgG (for most available antibodies)

• Detection:

  • Expected band: 100-140 kDa

  • Verify specificity using knockdown/knockout controls as demonstrated in published studies

What are the recommended protocols for immunohistochemistry using TMEM132A antibodies?

For successful TMEM132A immunohistochemistry:

• Tissue preparation:

  • Formalin-fixed paraffin-embedded (FFPE) sections

  • Fresh-frozen tissue sections are also compatible with some antibodies

• Antigen retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Optimization may be required for specific tissue types

• Antibody dilution:

  • Starting range: 1:250-1:1000

  • Titrate for optimal signal-to-noise ratio

• Controls:

  • Positive control: Mouse brain tissue

  • Negative control: Isotype antibody or TMEM132A knockout tissue

• Signal detection:

  • DAB substrate for brightfield applications

  • Fluorescent secondary antibodies for co-localization studies

How can I validate the specificity of TMEM132A antibodies?

Rigorous validation ensures experimental reliability:

• Genetic approaches:

  • siRNA knockdown in cell culture (validated in HeLa and HEK293 cells)

  • CRISPR/Cas9 knockout models

  • Analysis of Tmem132a knockout mouse embryonic fibroblasts (MEFs)

• Specificity tests:

  • Western blot showing appropriate molecular weight (100-140 kDa)

  • Reduced/absent signal in knockdown/knockout samples

  • Immunoprecipitation followed by mass spectrometry verification

• Immunostaining validation:

  • Co-localization with known markers (e.g., ER markers for TMEM132A)

  • Comparison of staining patterns across multiple antibodies targeting different epitopes

Example validation data from published research:

• TMEM132A knockdown efficiency can be assessed by qPCR and Western blot

• Protein levels in wild-type versus knockout MEFs show complete absence of the expected band in homozygous knockout samples

How does TMEM132A regulate the Wnt signaling pathway?

TMEM132A influences Wnt signaling through multiple mechanisms:

• Wnt ligand stabilization:

  • TMEM132A depletion decreases both cytoplasmic and secreted WNT3A and WNT5A protein levels

  • Cycloheximide chase experiments demonstrate increased Wnt ligand degradation in TMEM132A-silenced cells

• Enhancement of WLS-Wnt interaction:

  • TMEM132A physically interacts with WLS (Wntless), a key Wnt chaperone protein

  • Co-immunoprecipitation experiments confirm this interaction

  • Co-localization of TMEM132A and WLS occurs predominantly in the ER

• Regulation of LRP6 co-receptor:

  • TMEM132A physically interacts with and stabilizes LRP6, a critical Wnt co-receptor

  • TMEM132A downregulation destabilizes LRP6, diminishing Wnt signaling capacity

• β-Catenin activation:

  • In TMEM132A knockout MEFs, β-Catenin phosphorylation at Ser45 is increased and total β-Catenin levels are decreased

  • Expression of Wnt target genes (Axin2, Cyclin D1) is reduced in knockout cells

For experimental investigation of these mechanisms, dual-luciferase reporter assays with Topflash reporter and co-culture systems can effectively measure Wnt pathway activation .

What role does TMEM132A play in neural development and cell migration?

TMEM132A is crucial for neural development and cell migration:

• Neural tube development:

  • Tmem132a-/- mouse embryos exhibit ~85% penetrance of spina bifida

  • Mutant embryos show prenatal lethality

• Mesoderm migration:

  • TMEM132A knockout results in impaired lateral migration of the paraxial mesoderm in the posterior trunk

  • This migration defect contributes to neural tube closure failure

• Cellular migration mechanisms:

  • TMEM132A knockdown in HeLa and HEK293 cells causes:

    • Defective cell migration in scratch assays

    • Random, erratic cell movement trajectories

    • Thinner filopodium-like protrusions instead of normal lamellipodia

• Molecular pathways affected:

  • Reduced RhoA activation

  • Decreased phosphorylation of cofilin and MLC2

  • Altered actomyosin polymerization

  • Disrupted cytoskeletal remodeling

For studying these effects, recommended methodologies include:

• Scratch assays with time-lapse imaging

• Quantification of migration trajectories and cell morphology

• Immunoblotting for phosphorylation states of migration-related proteins

How can I design experiments to investigate TMEM132A's protein-protein interactions?

To study TMEM132A interactions with partners like WLS and LRP6:

• Co-immunoprecipitation approaches:

  • Forward approach: Immunoprecipitate tagged TMEM132A (e.g., Flag-tagged) and detect interacting partners by Western blot

  • Reverse approach: Immunoprecipitate partners (e.g., HA-tagged WLS) and detect TMEM132A

  • Controls: Include IgG control, input samples, and knockdown/knockout validation

• Subcellular co-localization:

  • Confocal microscopy with fluorescently-tagged constructs

  • Combined with organelle markers (e.g., ER-RFP)

  • Quantify co-localization using appropriate software and statistical analyses

• Tandem affinity purification with mass spectrometry:

  • Use cells with stable TMEM132A expression

  • Apply filter-aided sample preparation (FASP)

  • Identify novel interacting partners by mass spectrometry

• Functional validation:

  • Dual-luciferase reporter assays to measure Wnt pathway activation

  • Co-culture systems with signal-sending and signal-receiving cells

  • Rescue experiments in knockout cells with wild-type or mutant constructs

Example experimental design for co-IP from published research:

• Overexpression of Flag-tagged TMEM132A to co-IP endogenous WLS

• Overexpression of HA-tagged WLS to co-IP endogenous TMEM132A

• Co-expression of both tagged proteins to demonstrate bidirectional interaction

What methodological approaches can effectively study TMEM132A's role in integrin signaling and cytoskeletal remodeling?

To investigate TMEM132A's impact on integrin signaling and cytoskeleton:

• Integrin expression analysis:

  • Western blot to measure integrin protein levels in wild-type versus knockout/knockdown cells

  • Flow cytometry to quantify surface expression of integrins

  • qPCR to determine if effects occur at transcriptional or post-transcriptional levels

• Cytoskeletal dynamics assessment:

  • Live cell imaging of actin dynamics using fluorescent reporters

  • Immunostaining for cytoskeletal markers:

    • Phalloidin for F-actin

    • Phospho-cofilin

    • Phospho-MLC2

  • Quantification of lamellipodia/filopodia formation at wound edges

• Small GTPase activity measurements:

  • RhoA activation assays

  • Rac1/Cdc42 pull-down assays

  • Visualization using FRET-based biosensors

• Integrin pathway activation:

  • Western blot for phosphorylated focal adhesion kinase (FAK)

  • Analysis of downstream effectors such as Src, paxillin

  • Immunostaining for focal adhesion formation and turnover

• Rescue experiments:

  • Reintroduction of wild-type TMEM132A in knockout cells

  • Structure-function analysis using domain-specific mutants

  • Pharmacological rescue using pathway-specific activators

These methodological approaches, combined with appropriate controls and quantitative analyses, will provide comprehensive insights into TMEM132A's molecular functions in integrin-mediated cytoskeletal remodeling and cell migration.

What are common challenges when working with TMEM132A antibodies and how can they be addressed?

Several challenges may arise when using TMEM132A antibodies:

• High molecular weight detection issues:

  • Problem: Inefficient transfer of 100-140 kDa TMEM132A protein

  • Solution: Extend transfer time, reduce methanol concentration in transfer buffer, or use specialized transfer systems for high molecular weight proteins

• Multiple bands in Western blot:

  • Problem: Non-specific binding or detection of isoforms/degradation products

  • Solution: Optimize antibody dilution (1:2000-1:10000) , include proper controls, and validate with knockout/knockdown samples

• Weak signal in IHC:

  • Problem: Insufficient antigen retrieval or antibody concentration

  • Solution: Compare TE buffer pH 9.0 with citrate buffer pH 6.0 , extend antigen retrieval time, or adjust antibody concentration (1:250-1:1000)

• Background in immunofluorescence:

  • Problem: Non-specific binding

  • Solution: Increase blocking time/concentration, optimize antibody dilution, and include appropriate negative controls

How can I determine the optimal fixation method for TMEM132A immunostaining?

Fixation methods significantly impact TMEM132A detection:

• Paraformaldehyde fixation (recommended):

  • 4% PFA for 10-20 minutes at room temperature

  • Preserves protein epitopes while maintaining cellular morphology

  • Compatible with most TMEM132A antibodies for IF/ICC applications

• Methanol fixation:

  • May expose different epitopes than PFA fixation

  • Test with your specific antibody if PFA results are suboptimal

• Tissue fixation for IHC:

  • Formalin-fixed paraffin-embedded sections require appropriate antigen retrieval

  • TE buffer pH 9.0 is generally recommended, with citrate buffer pH 6.0 as an alternative

• Fresh-frozen tissue sections:

  • May preserve antigenicity better for certain applications

  • Fix briefly in acetone or PFA before immunostaining

Always perform comparative analyses to determine the optimal fixation method for your specific experimental system and antibody.

What are emerging research areas for TMEM132A function beyond Wnt signaling and cell migration?

Recent findings suggest several promising directions for TMEM132A research:

• Developmental biology:

  • Exploring TMEM132A's role in other embryonic developmental processes beyond neural tube formation

  • Investigating tissue-specific functions using conditional knockout models

• Neurodevelopmental disorders:

  • Given the neural tube defects in knockout mice, examining potential connections to human neurodevelopmental conditions

  • Exploring TMEM132A variants in patient populations

• Cancer biology:

  • Investigating TMEM132A's potential roles in tumor progression through:

    • Wnt pathway dysregulation

    • Altered cell migration capabilities

    • Effects on integrin signaling that might influence metastasis

• Protein quality control:

  • Further exploring TMEM132A's ER localization and potential roles in:

    • ER stress responses

    • Protein folding and trafficking

    • Cellular proteostasis

Each of these directions presents opportunities for novel experimental approaches and therapeutic target identification.

How might single-cell approaches advance our understanding of TMEM132A function?

Single-cell technologies offer powerful tools for TMEM132A research:

• Single-cell RNA sequencing:

  • Mapping cell type-specific expression patterns of TMEM132A during development

  • Identifying co-expressed gene networks to infer functional associations

  • Analyzing transcriptional consequences of TMEM132A deletion with single-cell resolution

• Single-cell proteomics:

  • Quantifying TMEM132A protein levels and post-translational modifications

  • Identifying cell-to-cell variability in TMEM132A-associated protein complexes

• Live-cell imaging:

  • Tracking TMEM132A dynamics during cell migration and division

  • Visualizing protein-protein interactions in real-time using FRET or BiFC

  • Correlating TMEM132A localization with cellular behaviors