TMEM192 Antibody

What is TMEM192 and why are antibodies against it important in research?

TMEM192 is a lysosome-resident transmembrane protein consisting of 271 amino acids in humans and 266 amino acids in mice, with these orthologs sharing approximately 78% sequence identity . The protein has a molecular mass of approximately 30.9 kilodaltons and features four transmembrane domains with both N- and C-termini facing the cytoplasm . TMEM192 has gained significant research importance due to its consistent localization in lysosomes, making antibodies against this protein valuable tools for lysosomal research, particularly in immunoprecipitation-based enrichment of lysosomes from various cell types and tissues . The protein forms homodimers interconnected by disulfide bridges, a structural feature that contributes to its stability and functionality in the lysosomal membrane . Antibodies targeting TMEM192 have revolutionized lysosomal research by enabling the development of methods like the "tagless LysoIP" approach, which allows for the isolation and analysis of lysosomes from clinical samples without requiring genetic modification of the target cells .

What are the primary applications for TMEM192 antibodies in research?

TMEM192 antibodies serve multiple experimental applications in research settings, with Western blotting (WB) being among the most common uses for detecting and quantifying TMEM192 protein expression in cell or tissue lysates . Immunofluorescence (IF) and immunohistochemistry (IHC) applications allow researchers to visualize the subcellular localization of TMEM192, confirming its presence in lysosomal compartments through co-localization studies with established lysosomal markers like LAMP-2 . ELISA techniques utilizing these antibodies enable quantitative analysis of TMEM192 in various experimental conditions . Perhaps most significantly, TMEM192 antibodies have enabled breakthrough applications in lysosomal immunoprecipitation (LysoIP), where they can selectively capture intact lysosomes from homogenized cell preparations, allowing subsequent proteomic, lipidomic, or metabolomic analysis of lysosomal contents . This application has particular value in studying lysosomal storage disorders and other diseases involving lysosomal dysfunction, as it allows researchers to profile lysosomal content from patient samples without requiring genetic modification .

How should researchers select the appropriate TMEM192 antibody for their specific experimental needs?

When selecting a TMEM192 antibody, researchers should first consider the intended application, as different antibodies may be optimized for specific techniques such as Western blotting, immunoprecipitation, or immunofluorescence . Species reactivity is a critical selection factor, with commercially available antibodies showing varied reactivity profiles across human, mouse, rat, and even zebrafish samples; researchers should verify that the antibody recognizes TMEM192 from their experimental species, as epitope recognition can vary significantly between species despite sequence homology . The epitope location should be carefully evaluated, as exemplified by the differential recognition patterns of TMEM192AB1 (recognizing C-terminal residues 235-250) versus TMEM192AB2 (recognizing residues 200-235), which resulted in significantly different lysosomal enrichment efficiencies . Researchers should also consider whether their experimental design requires a monoclonal or polyclonal antibody, with monoclonal antibodies offering higher specificity for a single epitope while polyclonal antibodies may provide stronger signal through recognition of multiple epitopes . Additional considerations include whether the antibody is conjugated to a detection tag (such as FITC) or unconjugated, which affects visualization strategies, and whether validation data exists for the specific application in question .

What validation steps should be performed when using a new TMEM192 antibody?

Comprehensive validation of a new TMEM192 antibody should begin with Western blot analysis using positive controls (cells known to express TMEM192) and negative controls (TMEM192-knockout cells if available), ensuring the antibody detects bands of the expected molecular weight (approximately 30.9 kDa for the full-length human protein) . Specificity testing through immunofluorescence or immunohistochemistry should demonstrate the expected lysosomal localization pattern through co-staining with established lysosomal markers like LAMP-1 or LAMP-2 . For antibodies intended for immunoprecipitation applications, researchers should validate the ability to efficiently capture TMEM192 from cell lysates, comparing results with appropriate controls such as mock immunoprecipitation using non-specific antibodies or BSA-conjugated beads . Cross-reactivity assessment across species should be performed if working with non-human models, as demonstrated in studies showing that antibodies against human TMEM192 did not recognize the murine orthologue despite 78% sequence identity, necessitating the development of species-specific antibodies . Additional validation steps may include testing for potential cross-reactivity with other proteins through mass spectrometry analysis of immunoprecipitated material, and confirming antibody performance under the specific experimental conditions that will be used in the intended research .

What types of controls should be included in experiments using TMEM192 antibodies?

Rigorous experimental design with TMEM192 antibodies requires multiple levels of controls to ensure valid interpretation of results. Positive controls should include samples known to express TMEM192, such as HEK293 cells exogenously expressing tagged TMEM192 protein, while negative controls should incorporate TMEM192-knockout cells or tissues when available, as demonstrated in studies using TMEM192-/- murine embryonic fibroblasts . For immunoprecipitation experiments, researchers should implement parallel mock immunoprecipitations using non-specific antibodies (such as pre-immune serum) or non-antibody proteins (such as BSA) conjugated to the same beads used for TMEM192 immunoprecipitation . When performing subcellular localization studies, co-staining with established organelle markers (particularly lysosomal markers like LAMP-1, LAMP-2, or LAMTOR1) is essential to confirm the expected lysosomal localization . For antigen competition assays, pre-incubating the antibody with excess purified TMEM192 protein or the peptide used for immunization can confirm specificity by demonstrating loss of signal. Technical controls should include secondary-antibody-only staining to assess background signal, and comparative analysis using alternative TMEM192 antibodies recognizing different epitopes to verify consistent results across detection methods .

How can TMEM192 antibodies be utilized for lysosomal immunoprecipitation (LysoIP)?

The tagless LysoIP method utilizing TMEM192 antibodies represents a significant advancement in lysosomal research, enabling the isolation of intact lysosomes from clinical samples without requiring genetic modification of the cells . To implement this technique, researchers should begin by selecting an appropriate TMEM192 antibody with demonstrated efficacy in immunoprecipitation; studies have shown that TMEM192AB1 (recognizing C-terminal residues 235-250) performs significantly better than TMEM192AB2 in lysosomal enrichment applications . The selected antibody must be covalently coupled to magnetic beads using standard cross-linking procedures to create a stable immunoaffinity matrix . Cell or tissue samples should undergo gentle homogenization using a ball bearing homogenizer in isotonic potassium phosphate-buffered saline (KPBS) supplemented with protease and phosphatase inhibitors, a critical step for maintaining lysosomal integrity during isolation . The homogenate is then incubated with the antibody-conjugated beads, allowing capture of TMEM192-containing lysosomes while minimizing non-specific binding . Following thorough washing steps, the immunoprecipitated lysosomes can be processed for downstream analyses including proteomics, lipidomics, or metabolomics, providing valuable insights into lysosomal content and potential disease biomarkers .

What are the key differences between traditional HA-tagged TMEM192 LysoIP and the tagless LysoIP method?

The traditional LysoIP approach relies on exogenous expression of TMEM192 protein containing three tandem HA epitopes at its C-terminus (LysoTag) in target cells or engineered mouse models, requiring genetic modification that limits its applicability to experimentally tractable systems . In contrast, the tagless LysoIP method employs antibodies recognizing endogenous TMEM192 protein, eliminating the need for genetic modification and enabling lysosomal enrichment from primary human cells, tissues, and clinical samples that cannot be genetically engineered . While the HA-tag-based approach typically yields higher enrichment efficiency due to the high affinity of HA antibodies and the presence of multiple epitopes, the tagless method still provides sufficient enrichment for meaningful analysis of lysosomal content . Proteomic analyses have confirmed that both methods enrich for lysosome-annotated proteins including LAMP1, LAMTOR1, TMEM55B, CTSC, CTSD, and GBA1, though the enrichment factors may differ between the two approaches . The tagless method has demonstrated particular value in clinical applications, such as identifying disease biomarkers in lysosomal storage disorders, where genetically modifying patient samples would be impractical or impossible . Researchers should consider that the tagless method may require optimization of homogenization and immunoprecipitation conditions for different tissue types due to varying expression levels of endogenous TMEM192 across tissues .

What analytical techniques can be combined with TMEM192 antibody-based lysosomal isolation?

The tagless LysoIP method using TMEM192 antibodies creates a versatile platform for multiple downstream analytical approaches to characterize lysosomal contents and function . Proteomic analysis of immunopurified lysosomes can be performed using mass spectrometry to identify and quantify the lysosomal proteome, revealing changes in protein composition associated with disease states or experimental conditions . Lipidomic approaches can be applied to analyze lysosomal membrane lipids and internal lipid content, including specialized lipids like bis(monoacylglycero)phosphate (BMP) that are enriched in lysosomes and often altered in lysosomal storage disorders . Metabolomic analysis of isolated lysosomes can identify small molecules and metabolites that accumulate within these organelles, providing insights into metabolic functions and dysfunctions . Enzyme activity assays performed on isolated lysosomes allow assessment of specific lysosomal enzyme functions under controlled conditions, avoiding interference from cytosolic enzymes that might be present in whole-cell lysates . Importantly, these analytical techniques can be applied to lysosomes isolated from patient samples, enabling biomarker discovery and monitoring of disease progression or treatment response in conditions like lysosomal storage disorders, where the method has demonstrated the ability to detect characteristic biomarkers like elevated BMP levels .

How can TMEM192 antibodies be used to study lysosomal storage disorders (LSDs)?

TMEM192 antibodies enable the application of tagless LysoIP for studying lysosomes isolated directly from patient samples, overcoming a significant limitation of previous approaches that required genetic modification . This methodology allows researchers to identify and monitor disease-specific biomarkers in LSDs, as demonstrated by studies showing characteristic accumulation of bis(monoacylglycero)phosphate (BMP) in lysosomes from Niemann-Pick disease type C (NPC) patients, with enrichment levels correlating with disease severity (4-30 fold higher compared to healthy controls) . By isolating intact lysosomes, researchers can comprehensively analyze the accumulated substrates specific to different LSDs, potentially revealing novel biomarkers for diagnosis, disease progression monitoring, or treatment response evaluation . The ability to perform comparative analyses between affected and unaffected tissues from the same patient provides valuable insights into tissue-specific manifestations of LSDs . Additionally, TMEM192 antibody-based approaches can be used to evaluate the efficacy of therapeutic interventions by monitoring changes in lysosomal content before and after treatment . For detailed mechanistic studies, researchers can compare lysosomes isolated from patient-derived cells with those from TMEM192-deficient mouse models of LSDs, though studies indicate that under basal conditions, TMEM192 deficiency alone does not result in lysosomal storage pathology, suggesting compensatory mechanisms that could themselves be therapeutic targets .

What protocols are recommended for Western blot detection of TMEM192?

For optimal Western blot detection of TMEM192, researchers should begin with proper sample preparation, lysing cells or homogenizing tissues in RIPA buffer supplemented with protease inhibitors to prevent degradation of the target protein . Due to the transmembrane nature of TMEM192, samples should not be boiled before loading unless specifically recommended by the antibody manufacturer, as excessive heat can cause aggregation of membrane proteins . Protein separation should be performed on 10-12% SDS-PAGE gels to provide optimal resolution around the expected molecular weight of TMEM192 (approximately 30.9 kDa for the full-length human protein) . Following transfer to nitrocellulose or PVDF membranes, blocking should be performed with 5% non-fat dry milk or bovine serum albumin in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature . Primary antibody incubation should follow the manufacturer's recommended dilution (typically 1:500 to 1:2000) in blocking buffer overnight at 4°C . After washing, HRP-conjugated or fluorescently-labeled secondary antibodies appropriate for the host species of the primary antibody should be applied at the recommended dilution (typically 1:5000 to 1:10000) for 1-2 hours at room temperature . For tissues known to exhibit TMEM192 processing, researchers should be prepared to detect both the full-length protein (approximately 30.9 kDa) and the processed fragment (approximately 17 kDa) .

What is the recommended approach for immunofluorescence detection of TMEM192?

Effective immunofluorescence detection of TMEM192 begins with proper fixation of cells using 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.1% Triton X-100 for 5 minutes to allow antibody access to intracellular structures while preserving the lysosomal morphology . Blocking should be performed with 5% normal serum from the same species as the secondary antibody in PBS for 30-60 minutes at room temperature to minimize non-specific binding . Primary TMEM192 antibody should be applied at the manufacturer's recommended dilution (typically 1:50 to 1:200 for immunofluorescence) in blocking buffer and incubated overnight at 4°C to ensure maximal specific binding . To confirm lysosomal localization, co-staining with established lysosomal markers such as LAMP-1 or LAMP-2 is essential, requiring sequential or simultaneous incubation with antibodies against these proteins . Appropriate fluorescently-labeled secondary antibodies should be selected based on the host species of the primary antibodies, ensuring compatibility for co-localization studies . For optimal visualization of the typically punctate lysosomal pattern, confocal microscopy is recommended over standard fluorescence microscopy . When interpreting results, researchers should look for co-localization of TMEM192 signal with lysosomal markers, appearing as yellow puncta in merged images, while being aware that even in wild-type cells, not all lysosomes may contain detectable levels of TMEM192 due to potential heterogeneity in expression across the lysosomal population .

How can researchers troubleshoot specificity issues with TMEM192 antibodies?

When encountering specificity issues with TMEM192 antibodies, researchers should first verify antibody quality by testing against positive controls (cells overexpressing TMEM192) and negative controls (TMEM192-knockout cells if available), which can quickly identify antibodies with poor specificity . Cross-reactivity with other proteins can be addressed by performing epitope mapping to identify the exact recognition sequence of the antibody, and then conducting BLAST searches to identify proteins with similar sequences that might lead to cross-reactivity . For antibodies showing unexpectedly multiple bands on Western blots, researchers should consider whether these represent different post-translationally modified forms of TMEM192, such as the processed 17 kDa fragment observed in murine tissues, or potential glycosylation variants . Species-specific issues can be significant, as demonstrated by the inability of human TMEM192 antibodies to recognize the murine orthologue despite 78% sequence identity; in such cases, researchers must obtain or develop species-specific antibodies . For immunofluorescence applications showing high background signal, optimization of blocking conditions (testing different blocking agents like BSA, normal serum, or commercial blocking solutions) and increasing the stringency of wash steps may improve specificity . When troubleshooting immunoprecipitation applications, researchers should compare the performance of different TMEM192 antibodies recognizing distinct epitopes, as demonstrated by the superior performance of TMEM192AB1 compared to TMEM192AB2 in lysosomal enrichment .

What are the best practices for optimizing TMEM192 antibody-based lysosomal isolation?

Optimizing TMEM192 antibody-based lysosomal isolation begins with selecting the most effective antibody for the application, with research demonstrating that antibodies recognizing the C-terminal region (residues 235-250) like TMEM192AB1 perform significantly better than those targeting other regions . The antibody coupling density to magnetic beads should be optimized, as excessive antibody loading can paradoxically reduce specificity due to steric hindrance, while insufficient loading reduces capture efficiency; typically, 5-10 μg of antibody per mg of beads represents a good starting point for optimization . Cell homogenization conditions require careful balancing to achieve sufficient disruption for lysosome release while maintaining organelle integrity, with ball bearing homogenizers providing more consistent results than Dounce homogenizers; the clearance should be adjusted based on cell type (typically 8-12 μm) . The homogenization buffer composition significantly impacts lysosomal stability during isolation, with isotonic potassium phosphate-buffered saline (KPBS) supplemented with protease and phosphatase inhibitors recommended to minimize organelle rupture and content leakage . Incubation conditions for the immunocapture step should be optimized for each sample type, with typical protocols using 30-60 minutes at 4°C with gentle rotation; longer incubation times may increase yield but risk compromising lysosomal integrity . Wash buffer composition and wash step number must be balanced to remove contaminants while preserving specific interactions, with typical protocols using 3-5 washes with buffer of the same composition as the homogenization buffer .

How can TMEM192 antibodies contribute to comparative studies across species?

TMEM192 antibodies designed for species-specific detection enable comparative studies of lysosomal biology across evolutionary lineages, though researchers must account for the 78% sequence identity between human and murine TMEM192, which is sufficient to cause significant epitope recognition differences requiring species-specific antibodies . By developing antibody panels recognizing TMEM192 orthologs from multiple species (human, mouse, rat, zebrafish), researchers can conduct comparative studies of lysosomal function and dysfunction across model organisms, translating findings between systems with greater confidence . The observed tissue-specific proteolytic processing of murine TMEM192, which generates a 17 kDa fragment in most tissues except liver, raises interesting evolutionary questions about whether similar processing occurs in human and other species' TMEM192, representing an area where species-comparative antibody studies could provide valuable insights . For translational research, the ability to compare lysosomal composition between patient samples and animal models of disease using species-specific TMEM192 antibodies helps validate the relevance of model systems and identify species-specific differences that might affect therapeutic development . In conservation biology and evolutionary studies, comparing TMEM192 structure, processing, and function across diverse species could provide insights into the evolution of the lysosomal system and its adaptation to different physiological demands .

What are the emerging applications of TMEM192 antibody-based research in disease studies?

The tagless LysoIP method enabled by TMEM192 antibodies has opened new avenues for studying lysosomal dysfunction in human diseases beyond classical lysosomal storage disorders . Neurodegenerative diseases including Alzheimer's, Parkinson's, and Huntington's diseases are increasingly linked to lysosomal dysfunction, and TMEM192 antibody-based isolation of lysosomes from patient brain tissue or iPSC-derived neurons could reveal disease-specific alterations in lysosomal content and function . Cancer research may benefit from analyzing lysosomes isolated from tumor tissues, as cancer cells often exhibit altered lysosomal positioning and function that contribute to therapy resistance and metastatic potential . Metabolic disorders like obesity and diabetes feature lysosomal stress that could be characterized in detail using TMEM192 antibody-based approaches to isolate lysosomes from adipose, liver, or pancreatic tissues . In inflammatory and autoimmune diseases, lysosomal function in immune cells plays crucial roles in antigen processing and presentation, areas where TMEM192 antibody-based isolation could provide mechanistic insights . The ability to monitor changes in lysosomal composition before and after therapeutic intervention using non-invasive clinical samples opens possibilities for developing lysosomal biomarkers of treatment response across multiple disease areas .

What complementary techniques can enhance TMEM192 antibody-based research?

Complementary techniques can significantly enhance the value of TMEM192 antibody-based research, with CRISPR/Cas9 genome editing providing a powerful approach for generating TMEM192-knockout cell lines or animal models that serve as essential negative controls for antibody validation and functional studies . Live-cell imaging using fluorescently tagged TMEM192 constructs complements antibody-based static imaging, allowing researchers to study the dynamics of lysosomal movement, fusion events, and interactions with other cellular compartments over time . Super-resolution microscopy techniques like STORM or STED can overcome the diffraction limit of conventional microscopy, providing nanoscale details of TMEM192 distribution within the lysosomal membrane when used with highly specific antibodies . Proximity labeling approaches such as BioID or APEX can be combined with TMEM192 as a lysosomal marker to identify proteins that transiently interact with lysosomes, providing information about the dynamic lysosomal interactome . Flow cytometry-based approaches using fluorescently-conjugated TMEM192 antibodies can enable high-throughput analysis of lysosomal characteristics in heterogeneous cell populations, potentially allowing sorting of cells based on lysosomal parameters . Single-cell sequencing technologies combined with TMEM192 antibody-based cell sorting could reveal cell-to-cell variation in lysosomal gene expression patterns within tissues, providing insights into the heterogeneity of lysosomal function in normal and disease states .