TMEM47 Antibody

What is TMEM47 and where is it primarily expressed?

TMEM47 (transmembrane protein 47) is a membrane-localized protein with a canonical length of 181 amino acid residues and a molecular mass of approximately 20 kDa in humans. Expression profiling has demonstrated significant TMEM47 presence in multiple tissues including adult brain, fetal brain, cerebellum, heart, lung, prostate, and thyroid . The protein is also known by alternative names including brain cell membrane protein 1 and transmembrane 4 superfamily member 10, which may appear in older literature . Expression patterns are conserved across species, with orthologous genes identified in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken models, making it suitable for comparative studies across evolutionary lines .

What are the primary applications of TMEM47 antibodies in research?

TMEM47 antibodies serve multiple experimental applications in molecular and cellular biology research. The predominant applications include enzyme-linked immunosorbent assay (ELISA), which allows for quantitative detection of TMEM47 in solution-based samples . Additionally, these antibodies are employed in Western blotting (WB) for molecular weight confirmation and semi-quantitative analysis, immunohistochemistry (IHC) for tissue localization studies, and immunofluorescence (IF) for subcellular localization analysis . For zebrafish-specific research, specialized antibodies have been developed that demonstrate cross-reactivity between human and zebrafish TMEM47 orthologs, enabling evolutionary and developmental studies .

How can I select the appropriate TMEM47 antibody for my specific research application?

Selection of the optimal TMEM47 antibody should be guided by several experimental considerations. First, determine your primary application (ELISA, WB, IF, IHC) as different antibodies may be optimized for specific techniques . Consider the conjugation requirements; non-conjugated antibodies are versatile for most applications, while specialized studies may benefit from pre-conjugated options such as HRP-conjugated (for enhanced sensitivity in ELISA), FITC-conjugated (for direct fluorescence detection), or biotin-conjugated (for signal amplification systems) .

Additionally, assess species reactivity based on your experimental model. While most commercial antibodies target human TMEM47, some offer cross-reactivity with mouse, rat, or zebrafish variants . For quantitative studies examining TMEM47 expression in chemoresistant cancer models, antibodies validated against HCC cell lines with varying levels of chemoresistance would be particularly valuable .

What are the optimal immunodetection methods for evaluating TMEM47 expression in tissue samples?

For robust detection of TMEM47 in tissue samples, a multi-modal approach is recommended. When performing immunohistochemistry, formalin-fixed paraffin-embedded sections should undergo antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) to maximize epitope exposure. For membrane proteins like TMEM47, Triton X-100 permeabilization (0.1-0.5%) is essential for antibody access to intracellular epitopes .

In clinical specimens, particularly those from TACE-treated HCC patients, correlating TMEM47 expression with treatment response requires careful methodological standardization . For research examining the relationship between TMEM47 and chemoresistance, parallel immunodetection of drug resistance markers (such as MDR1) is advisable to establish meaningful correlations . Dual immunofluorescence techniques can be particularly revealing when examining TMEM47 co-localization with tight junction proteins, given its established role in junction organization .

How should TMEM47 overexpression or knockdown experiments be designed for functional studies?

For functional characterization of TMEM47, both gain-of-function and loss-of-function approaches have proven informative. Overexpression studies should utilize lentiviral open reading frame (ORF) systems with fluorescent tags (such as GFP) to enable monitoring of transduction efficiency and protein localization . The study by Zhang et al. demonstrated successful TMEM47 overexpression using lentiviral ORF clones of monomeric GFP-tagged human TMEM47, with lentiviral particles produced via transfection into 293 cells using appropriate packaging systems .

For knockdown experiments, TMEM47-specific shRNA lentiviral transduction particles have shown efficacy in reducing TMEM47 expression . Experimental design should include appropriate controls (such as MISSION® TurboGFP control transduction particles) to account for non-specific effects of viral transduction . Expression validation should be performed at both mRNA level (via RT-qPCR) and protein level (via western blotting or immunofluorescence) to confirm successful manipulation of TMEM47 levels .

What considerations are important when analyzing TMEM47 in chemoresistance studies?

When investigating TMEM47's role in chemoresistance, several methodological considerations are critical. First, establish appropriate cellular models with defined resistance profiles, such as the MHCC97L/CisR and MHCC97L/CisR2 cisplatin-resistant cell lines, which can be developed through chronic incubation with increasing cisplatin concentrations (100 to 5,000 ng/ml over 12 months) .

Functional assays should include measurements of cellular response to treatment (e.g., apoptosis assays to detect caspase activation) and drug metabolism (assessment of genes involved in drug efflux and metabolism) . For clinical correlation, careful patient stratification based on treatment response is essential - for example, separating TACE-treated HCC patients into "response" (complete response) and "non-response" (without complete response) groups .

Quantitative assessment of TMEM47 expression in patient samples should be correlated with clinical parameters and treatment outcomes to establish clinical relevance . Multi-parametric analysis incorporating both TMEM47 expression and established chemoresistance markers (such as multi-drug resistance-associated protein 1) provides more comprehensive insights than single-marker approaches .

How does TMEM47 contribute to cell junction regulation and what experimental approaches best elucidate this function?

TMEM47 plays a significant role in regulating the morphology and assembly of tight junctions from adherens junctions in vertebrate cells . To investigate this function, researchers should employ high-resolution imaging techniques such as super-resolution microscopy or transmission electron microscopy, which can visualize junction ultrastructure. Co-immunoprecipitation studies can identify TMEM47 binding partners within junction complexes, while FRAP (Fluorescence Recovery After Photobleaching) analysis can assess junction dynamics in the presence or absence of TMEM47.

For mechanistic studies, researchers should examine TMEM47's effect on the localization of tight junction proteins through immunofluorescence co-localization studies . Time-lapse microscopy of fluorescently-tagged junction proteins in TMEM47-manipulated cells can reveal dynamic assembly processes. Additionally, transepithelial/transendothelial electrical resistance (TEER) measurements provide functional readouts of junction integrity that can be correlated with TMEM47 expression levels.

What is the current understanding of TMEM47's paradoxical roles in different cancer types?

TMEM47 demonstrates context-dependent functionality across cancer types, necessitating careful experimental design when studying its role in specific malignancies. In hepatocellular carcinoma, TMEM47 appears to promote chemoresistance, with expression levels positively correlating with cisplatin resistance and poor treatment response . Similarly, in breast cancer, TMEM47 overexpression is associated with metastatic potential and aggressive phenotypes .

Conversely, in malignant melanoma, TMEM47 has been identified as a potential tumor suppressor gene . This paradoxical behavior suggests tissue-specific regulatory mechanisms and protein interaction networks. To investigate these divergent roles, comparative proteomic analysis across cancer types can identify differential binding partners, while pathway enrichment analysis of TMEM47-correlated genes may reveal tissue-specific signaling contexts.

Chromatin immunoprecipitation sequencing (ChIP-seq) studies can elucidate transcriptional regulation of TMEM47 across cancer types, potentially explaining expression differences. Functional studies should be designed with careful consideration of cellular context, using appropriate tissue-specific models and controls.

How can TMEM47-mediated chemoresistance mechanisms be effectively targeted in experimental models?

Based on current understanding, targeting TMEM47-mediated chemoresistance requires a multi-faceted approach. Research has shown that inhibition of TMEM47 can significantly reduce cisplatin resistance in HCC cells by enhancing caspase-mediated apoptosis and suppressing cisplatin-induced activation of drug efflux and metabolism genes .

Experimental approaches should include combination therapy models, where TMEM47 inhibition (via shRNA or small molecule inhibitors) is paired with conventional chemotherapeutics like cisplatin . Molecular pathway analysis should focus on apoptotic mechanisms (caspase activation, PARP cleavage) and drug metabolism pathways (expression of ABC transporters and detoxification enzymes) .

For translational relevance, patient-derived xenograft (PDX) models from chemoresistant tumors offer an opportunity to evaluate TMEM47-targeting strategies in more clinically relevant systems. Monitoring both tumor response and toxicity profiles is essential for comprehensive assessment of therapeutic potential.

What are common challenges when using TMEM47 antibodies and how can they be addressed?

When working with TMEM47 antibodies, researchers may encounter several technical challenges. Membrane proteins like TMEM47 can be difficult to extract efficiently, potentially leading to inconsistent signal intensity. To address this, optimization of membrane protein extraction protocols using specialized buffers containing appropriate detergents (RIPA buffer supplemented with 1% NP-40 or 0.5% sodium deoxycholate) is recommended .

Non-specific binding is another common issue, especially in tissues with high endogenous peroxidase activity. This can be mitigated through thorough blocking steps (3-5% BSA or 5-10% normal serum matching the secondary antibody host species) and inclusion of appropriate negative controls (isotype controls, secondary-only controls, and tissue from TMEM47 knockout models when available) .

Variable TMEM47 expression across tissue types may necessitate application-specific optimization. For example, brain tissue (where TMEM47 is highly expressed) may require different antibody dilutions than tissues with lower expression levels . Titration experiments determining optimal antibody concentration for each tissue type are advisable before proceeding with full experimental protocols.

How should discrepancies between TMEM47 mRNA and protein expression data be interpreted?

Discrepancies between mRNA and protein expression levels for TMEM47 may reflect post-transcriptional regulatory mechanisms and should be interpreted carefully. When such discrepancies are observed, researchers should consider several potential explanations: microRNA-mediated suppression of translation, protein degradation pathways affecting TMEM47 stability, or issues with antibody sensitivity or specificity .

To address these issues methodologically, multiple detection approaches should be employed. For mRNA quantification, both RT-qPCR and in situ hybridization provide complementary data . At the protein level, both western blotting and immunohistochemistry/immunofluorescence should be performed to validate expression patterns .

When analyzing data from chemoresistance studies, temporal considerations are important; TMEM47 mRNA upregulation might precede detectable protein changes, particularly in the context of cisplatin-induced expression . Time-course experiments capturing both mRNA and protein dynamics can provide more comprehensive insights into regulatory mechanisms.

What controls are essential when studying TMEM47 in patient-derived samples?

When investigating TMEM47 expression in clinical specimens, rigorous controls are essential for reliable interpretation. For immunohistochemical analysis of patient-derived samples, include positive control tissues with known TMEM47 expression (brain, heart, lung) to verify antibody performance in each staining batch .

Technical negative controls should include isotype controls and secondary antibody-only controls to assess non-specific binding. Additionally, paired analysis of tumor and adjacent non-tumor tissue from the same patient provides internal control for tissue-specific effects and baseline expression levels .

In studies correlating TMEM47 with treatment response (such as TACE for HCC), patient stratification and careful documentation of treatment details are critical . Control groups should include treatment-naïve patients with comparable disease characteristics to distinguish treatment-induced changes from inherent tumor properties . Multivariate analysis accounting for clinical confounders (tumor stage, liver function, etc.) strengthens the validity of TMEM47-related observations.

What are promising approaches for developing more specific TMEM47-targeting strategies?

Development of next-generation TMEM47-targeting approaches presents several promising research avenues. Structure-based drug design targeting specific functional domains of TMEM47 could yield small molecule inhibitors with enhanced specificity compared to global knockdown approaches . This would require detailed structural characterization of TMEM47 using techniques such as cryo-electron microscopy or X-ray crystallography.

Therapeutic antibody development represents another promising direction, potentially yielding function-blocking antibodies that interfere with TMEM47's interactions with junction proteins or chemoresistance-associated partners . Epitope mapping studies would be essential to identify functionally relevant antibody binding sites.

For genetic approaches, CRISPR-Cas9 technology offers precise gene editing capabilities that could be applied to modify specific TMEM47 domains in experimental models . Additionally, targeted delivery systems (such as nanoparticles conjugated with TMEM47 antibodies) could improve therapeutic efficacy while minimizing off-target effects in complex tissues.

How can TMEM47 expression data be integrated into predictive models for chemotherapy response?

Integration of TMEM47 expression data into predictive models for chemotherapy response represents an important translational application. Multivariate predictive models should incorporate TMEM47 expression alongside established clinicopathological parameters and other molecular markers of drug resistance .

Machine learning approaches utilizing TMEM47 expression data from pre-treatment biopsies, combined with treatment outcomes data, could identify patterns predictive of therapeutic response, particularly for cisplatin-based therapies like TACE . Such models would require validation in independent patient cohorts through prospective studies.

Development of standardized scoring systems for TMEM47 expression in clinical specimens would facilitate consistent data interpretation across institutions. Quantitative image analysis methods applied to TMEM47 immunohistochemistry could reduce inter-observer variability and enhance reproducibility of expression assessment .

Integrating TMEM47 data with broader molecular profiling (genomics, transcriptomics, proteomics) could reveal complex signatures with superior predictive power compared to single-marker approaches.