TMEM63A Antibody

What is TMEM63A and why is it important in biomedical research?

TMEM63A (Transmembrane Protein 63A) is a mechanosensitive ion channel with critical physiological functions. Recent research has demonstrated its essential role in pulmonary surfactant secretion in alveolar type 2 epithelial (AT2) cells, where it facilitates lung expansion through mechanical stretch response . Additionally, TMEM63A is implicated in central nervous system (CNS) myelination, with mutations in the TMEM63A gene causing infantile hypomyelinating leukodystrophy 19 (HLD19) . The protein contains eleven transmembrane helices in a monomeric configuration, structurally similar to Arabidopsis thaliana OSCA proteins . Given its roles in both respiratory function and neurological development, TMEM63A represents an important target for basic and translational research.

How does TMEM63A structure relate to its function?

TMEM63A functions as a mechanosensitive ion channel with eleven transmembrane helices arranged in a monomeric configuration, as revealed by recent cryo-EM studies . The ion permeation pathway has been located within this monomeric structure, along with non-protein density resembling lipid . Functionally, TMEM63A responds to mechanical stimuli such as cellular stretch, particularly in AT2 cells where it localizes to the limiting membrane of lamellar bodies (LBs) . This localization facilitates surfactant and ATP release from LBs fused with the plasma membrane during cell stretch . In oligodendrocytes, TMEM63A is present at both the plasma membrane and on lysosomes, where it modulates myelin/myelin-associated protein production . Disease-associated TMEM63A variants from patients with HLD19 show impaired trafficking to the cell membrane, suggesting proper subcellular localization is critical for function .

What are the key considerations when selecting a TMEM63A antibody for research applications?

When selecting a TMEM63A antibody, researchers should consider multiple factors to ensure experimental success. First, determine the target epitope: antibodies targeting different regions (C-terminal, N-terminal, or specific amino acid sequences like AA 212-420 or AA 701-750) may show different specificities and applications . Second, consider the species reactivity needed: available antibodies show varied cross-reactivity with human, mouse, rat, rabbit, cow, and horse TMEM63A, with predictable reactivity percentages (e.g., 100% for human, 93% for mouse) . Third, evaluate the required application: some antibodies are optimized for Western blotting, while others work well for ELISA or immunofluorescence . Fourth, consider whether conjugated antibodies (HRP, FITC, or biotin) would benefit your experimental design for direct detection . Finally, assess the antibody's validation data, particularly focusing on published studies using the antibody in contexts similar to your planned experiments.

How should researchers validate a TMEM63A antibody before experimental use?

Thorough validation of TMEM63A antibodies is essential for research reliability. Begin with positive and negative controls: test the antibody in tissues or cell lines known to express TMEM63A (such as AT2 cells or oligodendrocytes) versus those with minimal expression or TMEM63A knockout models . For Western blotting validation, confirm the band appears at the expected molecular weight (~807 amino acids for human TMEM63A) and disappears in knockout samples. When validating for immunofluorescence, compare staining patterns with known TMEM63A subcellular localizations, such as lamellar body limiting membranes in AT2 cells or plasma membrane and lysosomal locations in oligodendrocytes . For more rigorous validation, consider using multiple antibodies targeting different epitopes to corroborate findings. Additionally, validate specificity through RNAi knockdown experiments where TMEM63A signal should decrease proportionally to knockdown efficiency. Document all validation experiments comprehensively, including antibody dilutions, incubation conditions, and detection methods.

What are the different types of TMEM63A antibodies available and their optimal applications?

TMEM63A antibodies are available in several configurations optimized for different experimental applications:

Researchers should select the antibody type based on their specific experimental needs, considering target epitope, detection method, and required species reactivity.

What are the best practices for using TMEM63A antibodies in Western blotting experiments?

For optimal Western blotting with TMEM63A antibodies, begin with careful sample preparation: use fresh tissue or cells and lyse in buffer containing protease inhibitors to preserve the intact protein. Given TMEM63A's transmembrane nature, use lysis buffers containing mild detergents like 1% Triton X-100 or RIPA buffer to effectively solubilize membrane proteins . During electrophoresis, use 8-10% SDS-PAGE gels to effectively resolve this large protein (~807 amino acids) . For transfer, consider using a wet transfer system with methanol-free transfer buffer to improve the transfer efficiency of this hydrophobic membrane protein. When blocking, 5% non-fat dry milk in TBST is generally effective, though BSA may provide lower background for some antibodies. For primary antibody incubation, start with the manufacturer's recommended dilution (typically 1:500 to 1:2000 for TMEM63A antibodies) and optimize as needed . Include positive controls (tissues known to express TMEM63A, such as lung tissue) and negative controls (knockout tissue if available). For detection, match your secondary antibody to the host species of your primary antibody (typically rabbit for most available TMEM63A antibodies) . If bands appear at unexpected molecular weights, consider the possibility of post-translational modifications, protein degradation, or splice variants.

How can researchers effectively use TMEM63A antibodies for immunofluorescence studies?

For successful immunofluorescence studies with TMEM63A antibodies, tissue fixation and permeabilization are critical first steps. For optimal results with this transmembrane protein, use 4% paraformaldehyde for fixation, followed by permeabilization with 0.2% Triton X-100 . When staining cultured cells, a shorter fixation time (10-15 minutes) may improve epitope accessibility. Given TMEM63A's presence in both plasma membrane and intracellular compartments like lysosomes , optimization of permeabilization conditions is crucial for balanced detection of all pools of the protein. For antibody incubation, start with the manufacturer's recommended dilution (typically 1:100 to 1:500 for IF applications) in a buffer containing 1-3% normal serum from the same species as the secondary antibody . Include appropriate controls: positive controls (tissues known to express TMEM63A such as oligodendrocytes for CNS studies or AT2 cells for lung studies) , and negative controls (no primary antibody, isotype control, or ideally TMEM63A knockout tissue). For co-localization studies, consider dual staining with markers for subcellular compartments of interest: LAMP1 for lysosomes or plasma membrane markers to distinguish between different TMEM63A pools . When using FITC-conjugated TMEM63A antibodies, be aware of potential photobleaching and optimize your imaging settings accordingly .

What methodologies are optimal for studying TMEM63A's mechanosensitive properties?

To study TMEM63A's mechanosensitive properties, researchers can employ several complementary methodologies. Patch-clamp electrophysiology represents the gold standard for directly measuring ion channel activity in response to mechanical stimuli. Using cell-attached or whole-cell configurations, researchers can apply negative pressure to the patch pipette or osmotic challenges to induce membrane stretch and record resulting currents . For studying TMEM63A in native contexts, ex vivo lung slice preparations coupled with calcium imaging using reporters like GCaMP6f have proven effective for monitoring stretch-induced calcium transients in AT2 cells . Alternative approaches include atomic force microscopy (AFM) to apply defined mechanical forces while simultaneously recording cellular responses. For high-throughput screening, automated patch-clamp systems can be combined with microfluidic devices that apply controlled shear stress or substrate stretching. When designing these experiments, researchers should include appropriate controls: TMEM63A knockout cells/tissues and pharmacological inhibitors of mechanosensitive channels. Additionally, complementary molecular approaches such as site-directed mutagenesis can help identify critical residues for mechanosensation by altering channel properties in response to mechanical stimuli.

How can researchers investigate the role of TMEM63A in pulmonary surfactant secretion?

To investigate TMEM63A's role in pulmonary surfactant secretion, researchers should employ a multi-pronged approach. First, establish appropriate model systems: primary AT2 cells, precision-cut lung slices, or transgenic mouse models with conditional TMEM63A deletion in AT2 cells using SPC-Cre recombinase . For analyzing surfactant secretion directly, measure phosphatidylcholine secretion using radiolabeled precursors or ELISA-based detection of surfactant proteins (SP-A, SP-B, SP-C, SP-D) in conditioned media following mechanical stretch stimulation . To visualize the process dynamically, implement live-cell imaging with lamellar body markers and fluorescently tagged TMEM63A to track fusion events during mechanical stimulation. Calcium imaging using indicators like GCaMP6f is essential for correlating mechanical stimulation, calcium transients, and surfactant release . For mechanistic insights, manipulate TMEM63A activity through genetic approaches (CRISPR/Cas9-mediated knockout or knockdown) or pharmacological tools, then assess effects on the surfactant secretion pathway. To establish physiological relevance, measure respiratory mechanics in control versus TMEM63A-deficient animals using techniques like forced oscillation or pressure-volume curve analysis . Finally, examine the TMEM63A-dependent transcriptome in AT2 cells using RNA-seq to identify potential downstream effectors in the surfactant secretion pathway.

What approaches should be used to study TMEM63A's role in CNS myelination disorders?

Studying TMEM63A's role in CNS myelination disorders requires specialized approaches spanning multiple scales of analysis. At the animal model level, use both constitutive and conditional TMEM63A knockout mice (particularly oligodendrocyte-specific deletions) to evaluate myelination deficits through histological, ultrastructural, and functional assessments . Zebrafish models provide complementary advantages for rapid screening and in vivo imaging of myelination processes . For human relevance, develop induced pluripotent stem cell (iPSC) lines from HLD19 patients carrying TMEM63A mutations and differentiate them into oligodendrocytes to study cell-autonomous defects in myelination capacity. At the cellular level, evaluate oligodendrocyte development and myelination using primary cultures and organoid systems, comparing wild-type to TMEM63A-deficient or mutant conditions . Assay for critical myelin proteins (MBP, PLP, MOG) using western blotting, qPCR, and immunostaining to quantify myelin production . For mechanistic insights, perform subcellular localization studies to track TMEM63A trafficking between plasma membrane and lysosomal compartments, particularly focusing on how disease-associated variants affect this distribution . Apply sophisticated imaging approaches like super-resolution microscopy to visualize TMEM63A's interaction with the myelination machinery. Finally, employ electrophysiological techniques to assess functional consequences of altered myelination in both ex vivo slice preparations and in vivo recordings in model organisms.

How can researchers investigate the structure-function relationship of TMEM63A using antibodies?

Investigating TMEM63A structure-function relationships using antibodies requires sophisticated approaches combining structural biology with functional assays. Researchers should begin by mapping the epitopes of available antibodies to specific domains within TMEM63A's eleven transmembrane helices structure . This allows strategic selection of antibodies that recognize functionally important regions. For conformational studies, use antibodies that preferentially bind active versus inactive channel states in immunoprecipitation or flow cytometry applications. Combine antibody-based approaches with site-directed mutagenesis of key residues in the ion permeation pathway or mechanical sensing domains, then use conformation-specific antibodies to detect resulting structural changes . For direct functional assessment, employ proximity ligation assays (PLA) with antibodies against TMEM63A and potential interaction partners to identify conformational changes during mechanical activation. To examine how patient mutations affect structure, use antibodies in pulse-chase experiments to track the biosynthesis, trafficking, and degradation of wild-type versus mutant TMEM63A . For higher resolution insights, complement antibody approaches with structural techniques like cryo-EM, using antibody fragments (Fabs) to stabilize specific conformations . Finally, for in situ structural analysis, apply advanced imaging techniques like FRET using labeled antibodies targeting different TMEM63A domains to monitor conformational changes in living cells during mechanical stimulation.

What are common challenges when working with TMEM63A antibodies and how can they be addressed?

Researchers working with TMEM63A antibodies face several common challenges. First, non-specific binding can occur due to the hydrophobic nature of this transmembrane protein. To address this, implement more stringent blocking (5% BSA instead of milk for Western blots) and increase washing steps (use 0.1% Tween-20 in TBS and wash 5×5 minutes) . Second, the epitope accessibility may be limited in fixed tissues due to TMEM63A's multiple transmembrane domains. Try alternative fixation methods (methanol instead of paraformaldehyde) or antigen retrieval techniques (citrate buffer pH 6.0, heat-mediated) . Third, different subcellular localizations (plasma membrane vs. lysosomes) may require optimized permeabilization conditions: use 0.1% saponin for selective plasma membrane permeabilization or 0.2% Triton X-100 for complete permeabilization . Fourth, variable expression levels across tissues may necessitate loading controls specific to the subcellular compartment where TMEM63A is being studied. Fifth, TMEM63A may undergo post-translational modifications affecting antibody recognition; validate antibodies against both native and denatured forms of the protein. Finally, for challenging applications like co-IP, consider crosslinking techniques to stabilize TMEM63A's membrane interactions before extraction.

How can researchers differentiate between TMEM63A and its close homologs (TMEM63B/C) in their experiments?

Differentiating between TMEM63A and its close homologs TMEM63B/C requires careful experimental design. First, select antibodies targeting unique regions with minimal sequence homology between family members; the C-terminal domain often shows greater divergence than transmembrane regions . Validate antibody specificity using overexpression systems with each family member individually expressed, then perform Western blots to confirm selective detection. For more rigorous validation, use knockout tissues or cells for each family member as negative controls . In immunostaining experiments, leverage the distinct tissue and subcellular expression patterns: TMEM63A and TMEM63B are co-expressed in AT1 and AT2 cells but at different levels, while their expression patterns in oligodendrocytes may differ . For transcript-level differentiation, design qPCR primers spanning unique exon junctions and validate their specificity against all three family members. Consider using RNA-seq data to identify tissues or developmental stages where one family member predominates. In functional studies, utilize the differential sensitivity to mechanical stimuli or ion selectivity that may exist between family members. Finally, for mechanistic studies, employ rescue experiments with individual family members in triple-knockout backgrounds to assess functional redundancy versus specificity.

What considerations are important when analyzing conflicting data from different TMEM63A antibodies?

When faced with conflicting data from different TMEM63A antibodies, researchers should implement a systematic troubleshooting approach. First, compare the epitopes targeted by each antibody: those recognizing different domains (N-terminal, C-terminal, or specific transmembrane regions) may detect different conformational states or post-translationally modified variants of TMEM63A . Second, evaluate each antibody's validation status through literature review and repository databases to assess reported specificity. Third, perform side-by-side validation experiments using positive controls (overexpression systems), negative controls (knockdown/knockout samples), and competition assays with the immunizing peptide. Fourth, consider whether discrepancies reflect biological reality: different subcellular pools of TMEM63A may have distinct properties, with plasma membrane versus lysosomal TMEM63A potentially differing in conformation or modification status . Fifth, assess technical variables such as fixation methods, antibody concentrations, incubation conditions, and detection systems that might contribute to discrepancies. Finally, implement alternative methods that don't rely on antibodies (such as CRISPR-Cas9 tagging of endogenous TMEM63A with fluorescent proteins) to provide independent verification. When reporting conflicting results, transparently document all antibodies used (including catalog numbers, lots, dilutions) and present parallel validation data to allow readers to assess reliability.