TMEM184C Antibody

What is TMEM184C and why is it significant for research?

TMEM184C (Transmembrane protein 184C, also known as TMEM34) is a multi-pass transmembrane protein with potential tumor suppressor functions that may play a significant role in cell growth regulation . The protein has gained research interest due to its possible involvement in cancer biology and cellular regulatory pathways . TMEM184C belongs to the transmembrane protein family, which generally functions in cellular communication, transport, and signaling across biological membranes. Understanding TMEM184C's function requires specific antibodies that can reliably detect and quantify this protein in various experimental contexts . The significance of TMEM184C in research extends to its potential role in tumorigenesis and cellular proliferation pathways, making it an important target for cancer studies and basic biological investigations.

What are the key structural and functional characteristics of TMEM184C antibodies?

TMEM184C antibodies are immunoglobulins specifically designed to recognize and bind to epitopes on the TMEM184C protein. Commercially available TMEM184C antibodies are typically polyclonal antibodies raised in rabbits against synthetic peptides corresponding to specific regions of the human TMEM184C protein . For instance, some antibodies target the C-terminal region, with epitopes located between amino acids 407-438 of human TMEM184C . Others may target regions near the carboxy terminus, such as within amino acids 340-390 . The antibodies undergo rigorous purification processes, including affinity chromatography purification via peptide columns or protein A columns followed by peptide affinity purification, to ensure high specificity and minimal cross-reactivity . These structural characteristics enable TMEM184C antibodies to function effectively in various research applications including Western blotting, ELISA, immunohistochemistry, and immunofluorescence analyses across human, mouse, and rat samples .

How does TMEM184C differ from other TMEM family proteins such as TMEM184B?

While TMEM184C and TMEM184B are both members of the transmembrane protein family, they exhibit distinct biological functions and tissue distributions. TMEM184C is primarily associated with cellular growth regulation and potential tumor suppressor activity , whereas TMEM184B has been implicated in neurodevelopmental processes . Recent research has identified pathogenic variants in TMEM184B that cause neurodevelopmental disorders including intellectual disability, corpus callosum hypoplasia, seizures, and microcephaly . TMEM184B contains a pore domain where disease-associated variants cluster and appears to influence synaptic structure and axon degeneration in evolutionarily conserved models . Unlike TMEM184C, TMEM184B has been specifically studied in neural contexts where it affects Wnt signaling pathways and potentially contributes to neuronal differentiation, migration, and survival . These differences highlight the importance of using specific antibodies that can distinguish between these related but functionally distinct proteins when conducting research in either cancer biology or neurodevelopmental fields.

What are the validated applications for TMEM184C antibodies in research?

TMEM184C antibodies have been validated for multiple research applications including Western blot (WB), ELISA (E), immunohistochemistry on paraffin-embedded tissues (IHC-P), and immunofluorescence (IF) . For Western blot applications, the recommended dilution is typically 1:2000, allowing for sensitive detection of TMEM184C protein in cell and tissue lysates . When used for immunohistochemistry, these antibodies are effective starting at concentrations of 5 μg/mL, enabling visualization of TMEM184C expression patterns in tissue sections . For immunofluorescence applications, higher concentrations starting at 20 μg/mL are recommended to achieve optimal signal-to-background ratios . ELISA applications utilize TMEM184C antibodies to quantitatively measure protein levels in solution, providing another dimension to protein expression analysis . These diverse applications make TMEM184C antibodies versatile tools for researchers investigating this protein's expression, localization, and function across various experimental systems and model organisms including human, mouse, and rat samples .

How should Western blot protocols be optimized for TMEM184C detection?

Optimizing Western blot protocols for TMEM184C detection requires careful consideration of several methodological aspects to ensure specific and sensitive results. Begin with proper sample preparation, using lysis buffers containing protease inhibitors to prevent TMEM184C degradation during extraction from tissues or cell lines. The predicted molecular weight of TMEM184C is approximately 50 kDa (50142 Da calculated) , which should guide gel percentage selection—typically 10-12% SDS-PAGE gels provide optimal resolution for this size range. During transfer, use PVDF membranes rather than nitrocellulose for transmembrane proteins like TMEM184C to ensure efficient protein binding and retention . Blocking should be performed with 5% non-fat dry milk or BSA in TBST for at least one hour at room temperature to minimize non-specific binding. For primary antibody incubation, use the recommended dilution of 1:2000 in blocking buffer, and optimize incubation conditions (typically overnight at 4°C) to maximize specific binding while minimizing background. Multiple washing steps with TBST should follow before and after secondary antibody incubation to remove unbound antibodies. Include appropriate positive controls (tissues known to express TMEM184C) and negative controls (tissues lacking TMEM184C expression or blocking peptide competition) to validate specificity of the detected bands .

What considerations are important for immunohistochemical detection of TMEM184C?

Immunohistochemical detection of TMEM184C requires specific methodological considerations to achieve reliable and interpretable results. First, tissue fixation and processing significantly impact antibody performance—formalin-fixed paraffin-embedded (FFPE) tissues require proper antigen retrieval methods to expose TMEM184C epitopes that may be masked during fixation . Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be empirically tested to determine optimal conditions for TMEM184C antibody binding. Starting with the recommended concentration of 5 μg/mL for IHC-P applications , researchers should perform antibody titration experiments to determine the optimal concentration that maximizes specific staining while minimizing background. Detection systems should be selected based on the required sensitivity, with avidin-biotin complex (ABC) or polymer-based systems generally providing good results for TMEM184C visualization. Counterstaining with hematoxylin helps contextualize TMEM184C expression within tissue architecture, while careful interpretation should consider the transmembrane nature of TMEM184C—expecting membranous or cytoplasmic staining patterns rather than nuclear localization. Including positive control tissues with known TMEM184C expression and negative controls (either tissues lacking TMEM184C or primary antibody omission) is essential for validating staining specificity and distinguishing true signal from background or artifacts .

How should researchers design experiments to study TMEM184C expression in cancer models?

Designing experiments to study TMEM184C expression in cancer models requires a comprehensive approach incorporating multiple methodologies and proper controls. Begin by establishing a hypothesis based on existing knowledge of TMEM184C as a potential tumor suppressor with roles in cell growth regulation . Select appropriate cancer cell lines and tissue samples representing different cancer types and stages, as TMEM184C expression may vary across cancer subtypes and progression states . Implement a multi-level analysis approach: first, assess TMEM184C mRNA expression using qRT-PCR with validated primer pairs spanning exon-exon junctions to ensure specificity; then confirm protein expression using Western blotting with recommended antibody dilutions (1:2000) and immunohistochemistry starting at 5 μg/mL to visualize expression patterns within tumor architecture. For functional studies, design knockdown experiments using siRNA or CRISPR-Cas9 approaches targeting TMEM184C, paired with overexpression studies using tagged constructs to assess the impact on cancer cell growth, migration, and survival. Include appropriate controls at each experimental stage: housekeeping genes for qRT-PCR normalization, loading controls for Western blots, non-targeting siRNAs for knockdown experiments, and empty vector controls for overexpression studies . Correlate TMEM184C expression levels with clinical parameters and patient outcomes using properly annotated tissue microarrays and available cancer databases to establish potential prognostic significance .

What controls should be included when validating TMEM184C antibody specificity?

Validating TMEM184C antibody specificity requires a systematic approach with multiple complementary controls to ensure reliable research outcomes. First, include positive control samples known to express TMEM184C, which may include specific cell lines or tissues with documented TMEM184C expression . Equally important are negative control samples where TMEM184C expression is absent or minimal, which helps establish the threshold for specific versus non-specific signals. Peptide competition assays provide a powerful specificity control, where pre-incubation of the antibody with the immunizing peptide (the synthetic peptide used to generate the antibody, typically between amino acids 407-438 or 340-390 of human TMEM184C) should abolish or significantly reduce the specific signal. Genetic validation controls using TMEM184C knockdown (siRNA or shRNA) or knockout (CRISPR-Cas9) cell lines provide the gold standard for specificity assessment—the antibody signal should be proportionally reduced or eliminated in these samples. For TMEM184C antibodies with cross-species reactivity to mouse and rat , include samples from multiple species to confirm the expected cross-reactivity patterns and check for any unexpected differences in binding patterns or molecular weight detection. Testing multiple antibodies targeting different epitopes of TMEM184C can provide convergent evidence of specificity, as concordant results from independently developed antibodies strongly support authentic target detection rather than cross-reactivity artifacts .

How can researchers effectively compare results from different lots of TMEM184C antibodies?

Comparing results from different antibody lots requires careful experimental design and standardization to ensure data consistency and reproducibility in TMEM184C research. First, establish a reference sample set of positive controls (tissues or cell lines with known TMEM184C expression) and negative controls (samples with minimal or no TMEM184C expression) that will be used consistently across lot comparisons . When transitioning to a new antibody lot, perform side-by-side experiments using both old and new lots on identical sample sets under identical experimental conditions, including the same protein amounts, incubation times, detection reagents, and imaging parameters. Quantitatively assess potential differences by measuring signal intensity, signal-to-noise ratio, and detection threshold across multiple experiments and biological replicates. If discrepancies between lots are observed, validate the new lot using orthogonal approaches such as RNA expression correlation, peptide competition assays, or testing in TMEM184C knockout/knockdown models . Maintain detailed records of lot numbers, dates of experiments, and performance metrics for each lot to build a database of antibody performance over time. Consider creating a large batch of standardized lysates or fixed samples that can be aliquoted and stored long-term at -80°C to serve as consistent reference materials for qualifying new antibody lots throughout a multi-year research project . This systematic approach ensures that observed experimental variations reflect true biological differences rather than technical artifacts introduced by antibody lot variability.

What are common troubleshooting strategies for weak or absent TMEM184C antibody signal?

When encountering weak or absent TMEM184C antibody signal, researchers should implement a systematic troubleshooting approach addressing sample preparation, antibody conditions, and detection parameters. First, confirm sample integrity and TMEM184C expression in your experimental system, as expression levels may vary across tissues and cell types—consider using positive control samples with known TMEM184C expression . For protein degradation issues, ensure complete protease inhibition during sample preparation and avoid repeated freeze-thaw cycles of antibodies, which can significantly reduce activity . If using paraffin-embedded tissues, optimize antigen retrieval methods by testing different buffers (citrate vs. EDTA) and conditions (microwave vs. pressure cooker) to effectively unmask TMEM184C epitopes. Antibody concentration may need adjustment—try increasing primary antibody concentration or extending incubation time (overnight at 4°C instead of 1-2 hours at room temperature) . For Western blotting specifically, ensure efficient protein transfer by checking transfer efficiency with reversible protein stains, and consider using PVDF rather than nitrocellulose membranes for transmembrane proteins like TMEM184C. Enhanced chemiluminescence (ECL) signal can be amplified by using higher sensitivity detection reagents or switching to more sensitive detection methods such as fluorescent secondary antibodies with infrared imaging systems . If all these approaches fail, the epitope recognized by your specific antibody might be modified, masked, or absent in your experimental conditions—consider testing another TMEM184C antibody recognizing a different epitope region .

How should researchers address non-specific binding when using TMEM184C antibodies?

Non-specific binding when using TMEM184C antibodies can compromise experimental interpretation but can be systematically addressed through multiple optimization strategies. Begin by optimizing blocking conditions—increase blocking agent concentration (from 3% to 5% BSA or milk) and extend blocking time (from 1 to 2 hours) to more effectively mask non-specific binding sites on membranes or tissues . Adjusting antibody dilution is critical—if using a 1:2000 dilution for Western blotting results in high background, try more dilute solutions (1:3000 or 1:5000) while extending incubation time to maintain specific signal . Increase the stringency and duration of washing steps by using higher concentrations of Tween-20 in wash buffers (0.1% to 0.3%) and performing additional wash cycles to remove unbound antibody more effectively. For immunohistochemistry applications starting at 5 μg/mL, include additional blocking steps with normal serum matching the species of the secondary antibody to reduce non-specific binding of the secondary antibody to endogenous immunoglobulins . Consider pre-adsorption of the primary antibody with tissues or cell lysates from species with low TMEM184C homology to remove cross-reactive antibodies while retaining TMEM184C-specific binding. If working with tissues rich in endogenous biotin (liver, kidney), use special blocking kits to neutralize endogenous biotin before applying avidin-biotin detection systems. Finally, confirm that observed non-specific bands are truly non-specific by performing peptide competition assays and comparing results with TMEM184C knockdown or knockout samples .

What are the best practices for TMEM184C antibody storage and handling to maintain activity?

Proper storage and handling of TMEM184C antibodies are critical for maintaining their activity and ensuring experimental reproducibility across extended research timelines. According to manufacturer guidelines, TMEM184C antibodies can be stored at 4°C for up to three months for ongoing experiments, but long-term storage should be at -20°C where they remain stable for up to one year . When preparing antibody aliquots, use sterile techniques and divide the stock into single-use volumes appropriate for your typical experiments to minimize repeated freeze-thaw cycles, as each cycle can significantly reduce antibody activity. Store antibody solutions in non-stick, low-protein-binding tubes to prevent antibody loss through surface adsorption. The buffer composition significantly impacts stability—TMEM184C antibodies are typically supplied in PBS containing 0.02-0.09% sodium azide as a preservative, which prevents microbial contamination during storage . When diluting antibodies for experiments, use freshly prepared buffers containing stabilizing proteins like BSA (0.5-1%) to maintain antibody conformation and activity. Avoid exposing antibodies to direct light, especially fluorophore-conjugated secondaries, and minimize exposure to extreme temperatures during shipping or handling. Document storage conditions, freeze-thaw cycles, and dates of reconstitution or dilution for each antibody lot to track potential activity loss over time. If activity diminishes despite proper storage, antibody re-purification methods or precipitation with ammonium sulfate can sometimes restore partial activity, though obtaining a new lot is often more reliable for critical experiments .

How can TMEM184C antibodies be utilized in studying protein-protein interactions?

TMEM184C antibodies offer powerful tools for investigating protein-protein interactions through multiple complementary approaches. Immunoprecipitation (IP) using TMEM184C antibodies allows researchers to pull down TMEM184C along with its interacting partners from cell or tissue lysates, followed by mass spectrometry analysis to identify the complete interactome . For studying specific suspected interactions, co-immunoprecipitation (co-IP) experiments can be designed where TMEM184C is immunoprecipitated using the antibody and Western blotting is performed to detect specific candidate interacting proteins. Proximity ligation assays (PLA) provide an advanced application where TMEM184C antibodies are combined with antibodies against potential interacting partners, generating fluorescent signals only when the two proteins are in close proximity (<40 nm), enabling visualization of interactions within intact cells with subcellular resolution. Chromatin immunoprecipitation (ChIP) may be relevant if TMEM184C interacts with DNA-binding proteins or is involved in transcriptional complexes, particularly given its potential tumor suppressor function . For live-cell studies, TMEM184C antibodies can be conjugated to quantum dots or other photostable fluorophores to track dynamic interactions using techniques such as fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS). When conducting these advanced studies, careful validation of antibody specificity becomes even more critical—peptide competition assays and TMEM184C-deficient controls should be included to ensure that observed interactions are specific rather than artifacts of antibody cross-reactivity .

What approaches can be used to investigate TMEM184C post-translational modifications?

Investigating TMEM184C post-translational modifications (PTMs) requires specialized experimental approaches leveraging the capabilities of TMEM184C antibodies combined with additional techniques. Phosphorylation, one of the most common PTMs, can be studied using phospho-specific TMEM184C antibodies if available, or through immunoprecipitation with general TMEM184C antibodies followed by detection with anti-phosphotyrosine, anti-phosphoserine, or anti-phosphothreonine antibodies . Mass spectrometry offers a comprehensive approach—immunoprecipitate TMEM184C using validated antibodies and perform LC-MS/MS analysis to identify specific modified residues and quantify modification stoichiometry. Glycosylation, potentially relevant for a transmembrane protein like TMEM184C, can be investigated by treating samples with specific glycosidases (PNGase F, Endo H) prior to Western blotting with TMEM184C antibodies to observe mobility shifts indicative of glycan removal. For studying ubiquitination or SUMOylation, immunoprecipitate TMEM184C and probe with anti-ubiquitin or anti-SUMO antibodies, or perform the reverse experiment by immunoprecipitating ubiquitinated proteins and probing for TMEM184C. Pulse-chase experiments combined with immunoprecipitation using TMEM184C antibodies can reveal the dynamics of PTM acquisition and turnover. To understand the functional consequences of identified PTMs, site-directed mutagenesis of modified residues followed by functional assays will be necessary, particularly focusing on how modifications might affect TMEM184C's potential tumor suppressor function or cell growth regulatory properties .

How can researchers integrate TMEM184C antibody-based techniques with genomic and transcriptomic approaches?

Integrating TMEM184C antibody-based techniques with genomic and transcriptomic approaches enables comprehensive multi-omics analysis of TMEM184C biology across different levels of cellular regulation. Begin by establishing correlations between TMEM184C protein expression (detected by antibody-based methods) and mRNA levels (measured by RNA-seq or qRT-PCR) across experimental conditions, cell types, or patient samples to identify potential post-transcriptional regulatory mechanisms . Chromatin immunoprecipitation sequencing (ChIP-seq) can be performed on transcription factors identified as differentially active in cells with altered TMEM184C expression to map the regulatory networks controlling TMEM184C transcription. For investigating TMEM184C's potential tumor suppressor function , combine TMEM184C immunohistochemistry data from patient samples with whole genome or exome sequencing to correlate protein expression patterns with genetic alterations in the TMEM184C gene or related pathways. RNA immunoprecipitation (RIP) using TMEM184C antibodies can reveal if TMEM184C protein interacts with specific RNA species, potentially uncovering unexpected roles in post-transcriptional regulation. CRISPR-Cas9 mediated knockout or knockdown of TMEM184C followed by RNA-seq analysis enables identification of genes and pathways regulated downstream of TMEM184C, while parallel proteomics analysis using TMEM184C antibodies for Western blotting can confirm changes at the protein level. For spatial context, combine single-cell RNA-seq data with immunofluorescence using TMEM184C antibodies to correlate transcriptional states with protein localization patterns in specific cell populations within heterogeneous tissues . These integrated approaches provide complementary perspectives on TMEM184C function that could not be achieved through any single methodology alone.