THEM4 Antibody

What is THEM4 and why is it significant in research?

THEM4, also known as CTMP (Carboxyl-Terminal Modulator Protein), functions as a negative regulator of PKB/Akt signaling at the plasma membrane and has the capacity to reverse the phenotype of v-Akt-transformed cells . The significance of THEM4 in research stems from its involvement in cancer biology, particularly in glioblastomas where hypermethylation of its promoter and transcriptional downregulation have been observed . Additionally, THEM4 has been identified as a broad-range, high-activity acyl-CoA thioesterase through bioinformatic analysis and subsequent in vitro testing . Recent research has established THEM4 as a mitochondrial protein whose overexpression correlates with increased mitochondrial membrane depolarization and enhanced caspase-3 and PARP cleavage, suggesting its important role in apoptotic pathways . Understanding THEM4's function provides insights into cellular regulatory mechanisms and potential therapeutic targets in cancer research.

What is the difference between observed and calculated molecular weight of THEM4?

The discrepancy between observed and calculated molecular weights of THEM4 represents an important consideration when validating antibody specificity. According to immunoblot analyses, THEM4 shows an observed molecular weight of approximately 68 kDa in human liver tissue lysates , while the calculated molecular weight based on amino acid sequence is only 27,130 Da (approximately 27 kDa) . Another source reports an observed molecular weight of 26 kDa, which aligns more closely with the calculated weight . This discrepancy between sources and between observed versus calculated weights likely reflects post-translational modifications of the protein. As noted in the technical documentation, "The additional higher molecular weight bands seen in the immunoblot may represent post-translationally modified TMEM4" . Researchers should be aware of these different molecular weight profiles when interpreting Western blot results to avoid misidentification of the target protein.

What species reactivity should be considered when selecting a THEM4 antibody?

When selecting a THEM4 antibody for research, species reactivity represents a critical parameter that determines experimental applicability. Currently available antibodies demonstrate reactivity across multiple mammalian species, particularly human, mouse, and rat models . This cross-species reactivity facilitates comparative studies and translational research between rodent models and human samples. The conservation of immunogenic epitopes across these species suggests evolutionary preservation of THEM4 structure and function. When designing experiments, researchers should verify the documented reactivity for their species of interest and consider performing preliminary validation studies if working with species not explicitly listed in the antibody specifications. Additionally, researchers should note that even within confirmed reactive species, tissue-specific expression patterns may influence antibody performance across different sample types.

What are the validated applications for THEM4 antibodies?

THEM4 antibodies have been validated for multiple experimental applications, providing researchers with versatile tools for protein detection and localization. The primary validated applications include Enzyme-Linked Immunosorbent Assay (ELISA), Western blotting (WB), immunohistochemistry on paraffin-embedded tissues (IHC-P), and immunofluorescence (IF) . For Western blot applications, THEM4 antibodies have been specifically validated using human liver tissue lysates, with recommended working concentrations of 1-2 μg/mL . For immunohistochemistry on paraffin-embedded sections, researchers should initiate optimization at 2.5 μg/mL, while immunofluorescence applications should begin at a higher concentration of approximately 20 μg/mL . Alternative recommendations suggest dilution ranges of 1:500-1:2000 for Western blotting and 1:20-1:200 for both IHC and IF applications . This variance in recommended concentrations emphasizes the importance of antibody titration and optimization for each specific application and experimental system to achieve optimal signal-to-noise ratio while minimizing background and non-specific binding.

How should THEM4 antibodies be stored and handled to maintain activity?

Proper storage and handling of THEM4 antibodies are essential for maintaining reagent integrity and experimental reproducibility. THEM4 antibodies can be stored at 4°C for up to three months without significant loss of activity . For long-term storage, maintaining antibodies at -20°C provides stability for up to one year . To prevent protein degradation and preserve epitope recognition capacity, researchers should aliquot antibodies before freezing to avoid repeated freeze-thaw cycles, which can significantly reduce antibody functionality . The typical storage buffer consists of PBS containing 0.02% sodium azide and, in some formulations, 50% glycerol as a cryoprotectant . Researchers should note that sodium azide, while functioning as an antimicrobial preservative, can inhibit horseradish peroxidase (HRP) activity, potentially interfering with certain detection systems. When working with THEM4 antibodies, allow them to equilibrate to room temperature before opening the vial to prevent condensation, which could introduce contaminants and accelerate degradation. Additionally, always handle antibodies using proper laboratory techniques to prevent introduction of proteases or other contaminants.

What controls should be included when using THEM4 antibodies in immunoassays?

When designing experiments with THEM4 antibodies, the inclusion of appropriate controls ensures reliable and interpretable results. For Western blot analysis, researchers should include positive controls such as human liver tissue lysates, which have been validated to express THEM4 . Additionally, incorporating a molecular weight ladder allows precise identification of the target band, particularly important given the discrepancy between calculated (27 kDa) and observed (68 kDa) molecular weights of THEM4 . Negative controls should include samples known not to express THEM4 or, ideally, THEM4-knockout tissues or cells. For antibody specificity verification, consider including a blocking peptide control, where the antibody is pre-incubated with the immunogen peptide before application to the sample . In immunohistochemistry and immunofluorescence experiments, include an isotype control (rabbit IgG at equivalent concentration) to assess non-specific binding . Additionally, secondary-antibody-only controls help identify background signal from non-specific secondary antibody binding. For comprehensive validation, particularly in novel applications or sample types, researchers may consider employing orthogonal detection methods or alternative antibodies targeting different epitopes of THEM4.

How does THEM4 antibody epitope selection affect experimental outcomes?

The selection of THEM4 antibody based on epitope recognition characteristics can significantly impact experimental outcomes and data interpretation. Current commercially available THEM4 antibodies are raised against synthetic peptides near the center of human THEM4, specifically within amino acids 60-110 . This epitope selection has several important implications. First, epitopes in this central region may be more accessible in denatured Western blot applications but potentially masked in native conformations, affecting performance across different applications. Second, post-translational modifications occurring near this region could interfere with antibody binding, explaining some of the variability observed in molecular weight detection . Third, sequence variations between species within this epitope region could affect cross-reactivity, though current antibodies successfully recognize human, mouse, and rat THEM4 . For researchers investigating specific functional domains of THEM4, such as its interaction with PKB/Akt or its thioesterase activity, antibodies targeting relevant domains should be selected. Additionally, researchers investigating potential splice variants or truncated forms of THEM4 should consider whether the antibody's epitope would be present in these alternative forms. When studying protein interactions or conformational changes, epitope accessibility in the native protein structure becomes a critical consideration that may necessitate alternative antibody selection or experimental approaches.

What are the methodological considerations for detecting THEM4 in mitochondria?

Detecting THEM4 in mitochondria requires specific methodological considerations due to its subcellular localization and the technical challenges of mitochondrial protein research. Since THEM4 has been identified as a mitochondrial protein involved in membrane depolarization and apoptotic processes , researchers should employ techniques that preserve mitochondrial integrity while enabling specific detection. For immunofluorescence approaches, optimization of fixation and permeabilization protocols is crucial; paraformaldehyde fixation (typically 4%) followed by permeabilization with 0.1-0.2% Triton X-100 often provides good results for mitochondrial proteins . Co-staining with established mitochondrial markers (e.g., MitoTracker dyes or antibodies against TOMM20 or COX IV) enables confirmation of mitochondrial localization through confocal microscopy. For biochemical approaches, researchers should consider mitochondrial fractionation protocols to enrich mitochondrial proteins before Western blot analysis, improving detection sensitivity. When studying the role of THEM4 in mitochondrial membrane depolarization, combining antibody-based detection with functional assays such as JC-1 or TMRE staining can provide correlative data between THEM4 expression and mitochondrial function. Additionally, super-resolution microscopy techniques may provide insights into the precise submitochondrial localization of THEM4, potentially clarifying its functional interactions within this organelle.

How can THEM4 antibodies be used to investigate its role in cancer progression?

THEM4 antibodies offer valuable tools for investigating the protein's role in cancer progression, particularly in glioblastomas where hypermethylation of the THEM4 promoter has been reported . To comprehensively study THEM4's role in cancer, researchers should implement a multi-faceted approach. Immunohistochemical analysis of tumor tissue microarrays using validated THEM4 antibodies at 2.5 μg/mL can establish expression patterns across tumor grades and correlate with patient outcomes . Western blot analysis of tumor lysates compared with matched normal tissue can quantify expression differences, while cell line studies can explore dynamic changes in THEM4 levels during cancer progression or in response to treatments. Given THEM4's role as a negative regulator of PKB/Akt signaling, co-immunoprecipitation experiments using THEM4 antibodies can reveal changes in its interaction with Akt in different cancer contexts . Researchers can also employ THEM4 antibodies in chromatin immunoprecipitation (ChIP) assays to investigate potential epigenetic mechanisms regulating its expression, particularly relevant given the reported hypermethylation of its promoter in glioblastomas. For functional studies, combining THEM4 antibody-based detection with assays for apoptosis, cell proliferation, and mitochondrial function can elucidate the consequences of altered THEM4 expression in cancer cells. Additionally, immunofluorescence co-localization studies can reveal changes in THEM4's subcellular distribution in cancer cells compared to normal cells, potentially identifying altered protein trafficking as a mechanism in cancer progression.

What strategies can address cross-reactivity issues with THEM4 antibodies?

Cross-reactivity represents a significant challenge when working with THEM4 antibodies, potentially leading to misinterpretation of experimental results. Several strategies can mitigate this issue. First, researchers should conduct comprehensive antibody validation using positive and negative controls, including THEM4 knockdown or knockout samples where possible . Second, titration experiments to determine optimal antibody concentration can reduce non-specific binding while maintaining specific signal detection. The recommended working dilutions of 1-2 μg/mL for Western blotting, 2.5 μg/mL for immunohistochemistry, and 20 μg/mL for immunofluorescence provide starting points for optimization . Third, blocking protocol optimization is crucial; consider using alternative blocking agents (BSA, casein, or commercial blockers) if traditional methods yield high background. Fourth, more stringent washing procedures can reduce non-specific binding; increasing wash volume, duration, or detergent concentration may improve signal-to-noise ratio. Fifth, for immunohistochemistry and immunofluorescence, antigen retrieval method optimization can enhance specific epitope accessibility while reducing non-specific binding. Finally, consider employing alternative detection systems; switching from colorimetric to fluorescent or chemiluminescent detection may provide better discrimination between specific and non-specific signals. When cross-reactivity persists despite these measures, researchers should consider alternative antibodies targeting different THEM4 epitopes or complementary detection methods like mass spectrometry to confirm protein identity.

How can inconsistencies in THEM4 molecular weight detection be resolved?

Resolving inconsistencies in THEM4 molecular weight detection requires systematic investigation of both biological and technical factors. The reported discrepancy between calculated (27 kDa) and observed (68 kDa) molecular weights suggests post-translational modifications or alternative protein processing . To address this issue, researchers should first ensure sample preparation consistency, including standardized lysis buffers and protein denaturation conditions. Employing gradient gels (4-20%) can improve resolution across a wide molecular weight range, helping to identify multiple bands or subtle mobility shifts. Including phosphatase and deglycosylation treatments of protein samples prior to electrophoresis can help determine if phosphorylation or glycosylation contributes to the higher observed molecular weight. Alternative sample preparation methods, such as urea-based lysis for improved protein denaturation, may resolve anomalous migration patterns caused by residual protein structure. When multiple bands are observed, peptide competition assays using the immunizing peptide can help identify which bands represent specific THEM4 detection. For definitive molecular weight verification, researchers should consider orthogonal approaches such as mass spectrometry analysis of immunoprecipitated protein or parallel detection with alternative antibodies targeting different THEM4 epitopes. Additionally, comparison of detection patterns across multiple cell types or tissues may reveal tissue-specific post-translational modifications or processing events that explain the molecular weight variations.

What are the best practices for antibody validation in THEM4 research?

Implementing rigorous antibody validation protocols ensures reliable and reproducible results in THEM4 research. A comprehensive validation approach should include multiple complementary strategies. First, perform application-specific validation for each experimental technique (Western blot, IHC, IF, ELISA) rather than assuming transferability of validation across methods . Second, include positive controls (human liver tissue for THEM4) and negative controls (isotype controls, secondary antibody only, and ideally THEM4-knockout samples) . Third, verify antibody specificity through peptide competition assays, where pre-incubation with the immunizing peptide should abolish specific signal . Fourth, conduct antibody titration experiments to determine optimal working concentrations; starting with manufacturer recommendations (1-2 μg/mL for WB, 2.5 μg/mL for IHC, 20 μg/mL for IF) and adjusting based on signal-to-noise ratio . Fifth, perform cross-platform validation by confirming consistent detection across multiple techniques (e.g., correlating Western blot results with immunofluorescence localization). Sixth, employ orthogonal validation using complementary methods such as mass spectrometry or RNA expression analysis to confirm protein identity and expression patterns. Seventh, document all validation procedures, including antibody source, catalog number, lot number, and experimental conditions, to ensure reproducibility . Finally, periodically revalidate antibodies, particularly when changing lots or when substantial time has elapsed since previous validation, as antibody performance may change over time or between lots.

How can bispecific antibody technology be applied to THEM4 research?

Bispecific antibody technology offers innovative approaches for investigating THEM4 function and potential therapeutic applications. Unlike conventional THEM4 antibodies that recognize a single epitope, bispecific antibodies can simultaneously bind THEM4 and a second target, enabling novel experimental and potentially therapeutic strategies . For mechanistic studies, researchers could develop bispecific antibodies targeting THEM4 and its interacting partners like PKB/Akt to investigate their spatial relationship and functional interactions in situ. These tools could help visualize transient protein complexes through proximity-based detection methods such as Förster resonance energy transfer (FRET) when combined with appropriate fluorophores. In therapeutic research contexts, bispecific antibodies linking THEM4 to immune cell receptors could potentially redirect immune responses toward cells with abnormal THEM4 expression or localization, similar to approaches used in multiple myeloma treatments . For technological development, researchers should consider adapting established bispecific antibody formats such as tandem single-chain variable fragments (scFvs) or diabodies for THEM4 targeting, with careful epitope selection to ensure dual binding capability without steric hindrance. When designing such advanced reagents, in silico modeling of antibody-antigen interactions followed by empirical validation through binding assays would optimize performance. Additionally, selection of appropriate expression systems (mammalian, insect, or cell-free) for bispecific antibody production requires careful consideration of glycosylation requirements and structural complexity.

What are the implications of THEM4 antibody specificity for phage display technology?

Phage display technology presents significant opportunities for developing highly specific THEM4 antibodies with customized binding properties. Recent advances in antibody selection methods employ phage display libraries containing diverse antibody variants, including those based on single naïve human V domains with systematically varied complementarity-determining regions (CDRs) . Applied to THEM4 research, this approach could generate antibodies with unprecedented specificity for particular conformational states, post-translational modifications, or protein-protein interaction interfaces of THEM4. Successful implementation requires careful antigen preparation; researchers should consider using both peptide fragments and full-length recombinant THEM4 protein as selection targets to obtain diverse binding specificities. The selection strategy should incorporate negative selection steps against related thioesterase family members to eliminate cross-reactive antibodies. Multiple rounds of selection with increasing stringency (through shorter binding times or more vigorous washing) can enrich for high-affinity binders. High-throughput sequencing of selected phage populations provides valuable information about antibody diversity and enrichment patterns, potentially identifying multiple unique binding solutions . For validation, researchers should employ the selected antibodies across multiple applications (ELISA, Western blot, IHC, IF) to comprehensively characterize their performance profiles. Additionally, computational modeling of antibody-antigen interactions, guided by experimental binding data, can further advance our understanding of epitope-paratope interactions and inform future antibody engineering efforts for enhanced THEM4 detection specificity.

How can THEM4 antibodies contribute to understanding mitochondrial dysfunction in disease?

THEM4 antibodies represent valuable tools for investigating mitochondrial dysfunction in various diseases, given THEM4's established role in mitochondrial function and apoptotic pathways . To leverage these reagents effectively, researchers should implement multi-parametric approaches combining antibody-based detection with functional mitochondrial assays. Immunofluorescence co-localization studies using THEM4 antibodies alongside markers for mitochondrial subcompartments can reveal changes in THEM4 distribution during disease progression or in response to stressors. In neurodegenerative disorders, where mitochondrial dysfunction is a common feature, researchers can employ THEM4 antibodies in brain tissue sections to correlate THEM4 expression or localization with disease markers and patient outcomes. For metabolic diseases, combining THEM4 immunodetection with metabolic flux analysis can elucidate relationships between THEM4 levels and alterations in mitochondrial metabolism. Live-cell imaging approaches using fluorescently-labeled THEM4 antibody fragments could enable real-time visualization of THEM4 dynamics during mitochondrial stress responses. In cancer research, where THEM4 has already been implicated through its promoter hypermethylation in glioblastomas , antibody-based approaches can reveal whether mitochondrial THEM4 localization or function is altered across different cancer types or stages. Additionally, researchers investigating drug-induced mitochondrial toxicity could employ THEM4 antibodies to determine whether changes in THEM4 expression or localization serve as early indicators of mitochondrial stress, potentially contributing to the development of biomarkers for drug safety assessment.