ELAC2 Antibody

What is ELAC2 and why is it important in scientific research?

ELAC2 (elaC homolog 2) is a protein encoded by the HPC2/ELAC2 gene that functions as a zinc phosphodiesterase with tRNA 3'-processing endonuclease activity. This protein plays a critical role in tRNA maturation by removing 3'-trailers from precursor tRNAs . Importantly, ELAC2 has been identified as a potential prostate cancer susceptibility gene, making it particularly significant in cancer research . This protein has a calculated molecular weight of 92 kDa and is widely expressed throughout the body, with particularly high expression in heart, placenta, liver, skeletal muscle, kidney, pancreas, testis, and ovary tissues . ELAC2 is primarily localized in mitochondria but has also been detected in the nucleus, suggesting multiple functional roles depending on subcellular location .

What applications are ELAC2 antibodies validated for?

ELAC2 antibodies have been validated for multiple experimental applications, providing researchers with flexibility in their investigation approaches. The most common applications include Western Blot (WB), with validated dilution ranges of 1:200-1:1000 for some antibodies and up to 1:5000-1:50000 for higher-affinity recombinant versions . Additionally, these antibodies have been validated for Immunohistochemistry (IHC) at dilutions of approximately 1:50-1:500 , Immunofluorescence (IF) , Flow Cytometry (FC) for intracellular detection , Enzyme-Linked Immunosorbent Assay (ELISA) , and Immunoprecipitation (IP) . Researchers should note that optimal dilutions may vary between specific antibody products and experimental conditions, making protocol optimization essential .

What are the key species reactivity considerations for ELAC2 antibodies?

Species reactivity is a crucial consideration when selecting an ELAC2 antibody. Based on validation data, different commercially available antibodies show varied species reactivity profiles. For instance, Proteintech's polyclonal antibody (10071-1-AP) has demonstrated reactivity with human, mouse, and rat samples , making it versatile for cross-species research. In contrast, Proteintech's recombinant antibody (83468-6-RR) has been specifically validated for human samples . The NBP3-12934 antibody has validated reactivity with human samples and predicted reactivity with chimpanzee (98% sequence homology) . Researchers should carefully match antibody species reactivity to their experimental model system to ensure reliable results and may need to empirically test reactivity in species not explicitly validated by manufacturers .

How should ELAC2 antibodies be stored for optimal stability?

Proper storage of ELAC2 antibodies is essential for maintaining their functionality and extending their usable lifespan. According to product specifications, these antibodies should typically be stored at -20°C, where they remain stable for approximately one year after shipment . The antibodies are generally supplied in PBS buffer (pH 7.3) containing preservatives such as 0.02% sodium azide and 50% glycerol or Proclin 300 (0.025%) . Some formulations may also contain BSA for stability . While aliquoting is generally unnecessary for -20°C storage, it may be advisable for frequently used antibodies to minimize freeze-thaw cycles, which can degrade antibody quality over time . Always centrifuge antibody vials briefly before opening to collect the solution at the bottom of the tube .

What are the optimal protocols for Western blot detection of ELAC2?

For optimal Western blot detection of ELAC2, researchers should consider several critical parameters based on the protein's characteristics. ELAC2 has an observed molecular weight of approximately 92-97 kDa , requiring appropriate gel concentration selection (typically 7.5-10% SDS-PAGE) . For sample preparation, whole cell extracts from various cell lines including HEK-293, HeLa, MCF-7, Raji, and DU 145 have been successfully used for ELAC2 detection . The recommended antibody dilutions vary significantly between products, ranging from 1:200-1:1000 for conventional polyclonal antibodies to 1:5000-1:50000 for high-affinity recombinant antibodies .

For enhanced specificity, antigen retrieval may be necessary when working with tissue samples. Validated methods include using citrate buffer (pH 6.0) or TE buffer (pH 9.0) for 15 minutes . When troubleshooting, researchers should pay particular attention to blocking conditions and consider using 5% non-fat milk or BSA in TBST for blocking and antibody dilution. Secondary antibody selection should match the host species (typically rabbit IgG for commercial ELAC2 antibodies) . For verification of results, appropriate positive control cell lines include DU 145, HeLa, and HepG2 cells, which have been validated to express detectable levels of ELAC2 .

How can researchers validate the specificity of ELAC2 antibodies?

Validating ELAC2 antibody specificity is essential for ensuring reliable experimental results. A comprehensive validation approach should incorporate multiple complementary strategies. First, researchers should perform knockdown/knockout validation using siRNA, shRNA, or CRISPR-Cas9 targeted to ELAC2, followed by Western blot analysis to confirm a corresponding reduction in the detected protein band . Published literature has documented successful knockdown validation of certain ELAC2 antibodies .

Researchers should also perform immunoprecipitation experiments to confirm that the antibody can specifically isolate the ELAC2 protein from complex mixtures. For instance, immunoprecipitation of ELAC2 from Jurkat whole cell extracts using NBP3-12934 antibody has been successfully demonstrated . Additionally, peptide competition assays can be conducted using the immunizing antigen to block antibody binding, which should eliminate or significantly reduce signal if the antibody is specific.

Cross-validation using multiple antibodies targeting different epitopes of ELAC2 provides another layer of confirmation. If different antibodies detect the same protein band at the expected molecular weight (~92 kDa), this increases confidence in specificity . Finally, correlation of protein detection with known expression patterns (e.g., higher expression in heart, placenta, liver, skeletal muscle, kidney, pancreas, testis, and ovary) can provide additional validation .

What are the specific considerations for immunohistochemical detection of ELAC2?

Immunohistochemical detection of ELAC2 requires careful attention to several technical parameters for optimal results. Antigen retrieval is particularly critical; validated protocols recommend using either TE buffer at pH 9.0 or citrate buffer at pH 6.0 for 15 minutes . For paraffin-embedded sections, deparaffinization and rehydration must be complete before proceeding with antigen retrieval. Antibody dilutions typically range from 1:50-1:500 for IHC applications, but this should be experimentally optimized for each specific tissue type and fixation method .

When interpreting ELAC2 staining patterns, researchers should expect both cytoplasmic and nuclear localization, with predominant mitochondrial staining reflecting the protein's main subcellular location . Validated positive control tissues include mouse testis tissue and human breast carcinoma samples . To confirm staining specificity, negative controls should include omission of primary antibody and, ideally, tissues from ELAC2 knockout models.

For dual or multi-staining experiments, researchers should consider combining ELAC2 detection with mitochondrial markers to confirm the subcellular localization pattern. Due to potential background or non-specific staining, signal amplification methods like tyramide signal amplification may be beneficial for detecting low-abundance expression in certain tissues. Quantification of ELAC2 expression in tissue samples should ideally employ digital image analysis with appropriate normalization to account for variations in tissue processing and staining intensity .

How can researchers study ELAC2's differential localization between mitochondria and nucleus?

Investigating ELAC2's differential localization between mitochondria and nucleus requires specialized subcellular fractionation techniques combined with appropriate detection methods. Since ELAC2 is primarily mitochondrial but also present in the nucleus , researchers should employ a sequential fractionation protocol that efficiently separates these compartments while minimizing cross-contamination. Commercially available fractionation kits can be used, but careful validation of fraction purity using compartment-specific markers (e.g., VDAC1 for mitochondria, Lamin B1 for nucleus) is essential.

For immunofluorescence detection, co-staining with MitoTracker dyes or mitochondrial proteins like COX IV alongside nuclear counterstains can visualize the relative distribution between compartments. Super-resolution microscopy techniques such as STED or STORM can provide enhanced resolution of ELAC2's precise localization within these structures. When performing Western blot analysis on subcellular fractions, researchers should load equivalent amounts of protein from each fraction and normalize ELAC2 signals to appropriate loading controls for each compartment.

To investigate the functional significance of this dual localization, researchers might employ mutations in ELAC2's mitochondrial targeting sequence or nuclear localization signals, followed by phenotypic analysis. Additionally, inducing changes in mitochondrial stress or nuclear gene expression might reveal conditions that alter the distribution between compartments. Time-course experiments during cellular differentiation or in response to specific stimuli could further elucidate the dynamic regulation of ELAC2's subcellular trafficking .

What experimental approaches can address ELAC2's role in cancer biology?

Investigating ELAC2's role in cancer biology, particularly its connection to prostate cancer susceptibility , requires multi-faceted experimental approaches. Researchers should begin with comprehensive expression profiling across cancer cell lines and patient-derived samples, comparing ELAC2 levels between normal and cancerous tissues using both Western blot and immunohistochemistry. The antibody dilutions of 1:200-1:1000 for Western blot and 1:50-1:500 for IHC have been validated for such analyses .

Functional studies should employ knockdown or knockout strategies using siRNA, shRNA, or CRISPR-Cas9 technology targeted against ELAC2, followed by assessments of cellular phenotypes including proliferation, migration, invasion, and apoptosis. Published studies have successfully utilized ELAC2 knockdown approaches , providing methodological precedents. For mechanistic investigations, researchers should explore ELAC2's tRNA processing activity in cancer contexts, potentially using tRNA sequencing to identify altered tRNA maturation patterns in cells with manipulated ELAC2 levels.

Genetic association studies can further explore the relationship between ELAC2 variants and cancer risk or progression. Of particular interest are the missense mutations (Ser217Leu and Ala541Thr) originally associated with hereditary prostate cancer . Additionally, researchers might investigate potential therapeutic approaches targeting ELAC2, including the development of small molecule inhibitors of its enzymatic activity or the identification of synthetic lethal interactions in cancer cells with altered ELAC2 function. Combining these approaches with patient outcome data can establish the clinical relevance of ELAC2 as a biomarker or therapeutic target in various cancer types .

How can researchers resolve inconsistent ELAC2 detection results between different antibodies?

Inconsistent detection results between different ELAC2 antibodies can arise from several factors that require systematic troubleshooting. First, researchers should examine the epitope information for each antibody; different commercial antibodies target distinct regions of ELAC2, such as fusion proteins encompassing central regions or specific immunogens like Ag0105 . Epitope differences can lead to variable detection efficiency, especially if post-translational modifications, protein interactions, or conformational changes mask certain epitopes.

Researchers should compare the validated applications for each antibody, as some may be optimized for specific techniques but not others. For example, while 10071-1-AP is validated for WB, IF, IHC, and ELISA , 83468-6-RR shows high sensitivity in Western blot but is primarily validated for flow cytometry and ELISA for other applications . Additionally, examine species reactivity carefully; cross-species applications may show reduced sensitivity if the epitope region has sequence variations.

When antibodies show discrepant results, consider performing validation experiments such as knockdown/knockout controls with each antibody. This can help determine which antibody provides the most specific detection. Alternatively, use multiple antibodies in parallel, with coincident detection providing stronger evidence of true ELAC2 identification. Finally, adjust experimental conditions (fixation methods, antigen retrieval, blocking reagents) systematically for each antibody, as optimal conditions may vary significantly between antibody clones. Document these optimization efforts methodically to establish reliable detection protocols for each research context .

What controls should be implemented when studying ELAC2 in different experimental systems?

Implementing appropriate controls is essential for rigorous ELAC2 research across different experimental systems. For Western blot analysis, researchers should include positive control samples from validated cell lines such as DU 145, HeLa, HepG2, MCF-7, or Raji cells, which are known to express detectable levels of ELAC2 . Loading controls appropriate to the subcellular compartment being studied are essential, with mitochondrial markers like VDAC or COX IV when focusing on mitochondrial ELAC2, and nuclear markers like Lamin B1 when examining nuclear fractions.

For genetic manipulation studies, include both negative controls (non-targeting siRNA/shRNA or empty vector) and positive controls (targeting a gene with well-characterized knockdown phenotypes). When using ELAC2 antibodies for immunostaining, prepare technical negative controls by omitting primary antibody and biological negative controls using tissues or cells known to express minimal ELAC2. For functional studies, consider species-specific differences in ELAC2 activity; human ELAC2 shares high homology with chimpanzee (98%) but may have functional differences from more distant orthologs.

In disease-focused research, particularly cancer studies, include appropriate matched normal-tumor sample pairs from the same patients or tissues to control for individual variation. When examining ELAC2 variants, create isogenic cell lines differing only in the ELAC2 variant of interest to isolate its specific effects. Finally, for tRNA processing studies, include control tRNA substrates without ELAC2 recognition sites to confirm processing specificity. These comprehensive controls help distinguish true ELAC2-related effects from experimental artifacts across diverse research contexts .

How can researchers optimize ELAC2 detection in flow cytometry experiments?

Optimizing ELAC2 detection in flow cytometry requires attention to several key parameters, particularly given its predominant intracellular localization. For intracellular staining, researchers should use validated antibodies such as Proteintech's 83468-6-RR, which has been specifically tested for this application at a recommended concentration of 0.25 μg per 10^6 cells in a 100 μl suspension . Effective cell fixation and permeabilization are critical; protocols using 4% paraformaldehyde for fixation followed by permeabilization with 0.1-0.5% saponin or Triton X-100 have proven effective for intracellular protein detection.

To minimize non-specific binding, implement thorough blocking steps using 2-5% BSA or FBS in PBS before antibody incubation. Since ELAC2 has dual localization in mitochondria and nucleus , co-staining with compartment-specific markers can help differentiate subpopulations based on relative protein distribution. A431 cells have been validated as positive controls for flow cytometry detection of ELAC2 , making them useful for protocol optimization and as experimental references.

When analyzing flow cytometry data, compare ELAC2 signal between experimental conditions using both median fluorescence intensity and percentage of positive cells. Gating strategies should account for cell size, granularity, and viability to exclude debris and dead cells, which often show non-specific antibody binding. For multiparameter analysis, careful compensation is necessary when combining ELAC2 staining with other fluorescent markers. Finally, consistency in instrument settings between experiments is essential for reliable quantitative comparisons of ELAC2 expression across different conditions or cell types .

What are the key considerations for designing ELAC2 knockdown or knockout validation experiments?

Designing rigorous ELAC2 knockdown or knockout validation experiments requires careful planning and appropriate controls. When using RNA interference approaches (siRNA/shRNA), researchers should design at least 3-4 independent targeting sequences against different regions of ELAC2 mRNA to minimize off-target effects. Previous publications have successfully employed ELAC2 knockdown techniques , providing methodological precedents. For CRISPR-Cas9 knockout, design multiple guide RNAs targeting early exons or critical functional domains of ELAC2, and validate editing efficiency through sequencing.

Regardless of the gene silencing approach, comprehensive validation of knockdown/knockout efficiency is essential at both mRNA level (using qRT-PCR) and protein level (using Western blot with validated antibodies at dilutions of 1:200-1:1000) . When interpreting phenotypes, consider ELAC2's dual localization in mitochondria and nucleus , as perturbation of different pools might yield distinct effects. Additionally, create rescue experiments by reintroducing wild-type ELAC2 or specific variants to confirm phenotype specificity.

For functional validation, assess tRNA processing directly, as ELAC2 plays a key role in removing 3'-trailers from precursor tRNAs . Analyze mitochondrial function parameters like ATP production, membrane potential, and respiration rates, given ELAC2's predominant mitochondrial localization . Finally, examine cell viability, proliferation, and cancer-related phenotypes, particularly in prostate cancer models, considering ELAC2's proposed role in cancer susceptibility . These comprehensive approaches ensure robust validation of ELAC2's functional roles across different biological contexts.

What emerging technologies might advance ELAC2 research beyond current antibody-based methods?

Emerging technologies have significant potential to complement and extend traditional antibody-based ELAC2 research methods. CRISPR-based tagging systems, such as CRISPR-Cas9 knock-in of fluorescent proteins or epitope tags at the endogenous ELAC2 locus, can enable live-cell imaging and functional studies without relying on antibodies. This approach eliminates concerns about antibody specificity while maintaining physiological expression levels. Similarly, proximity labeling techniques like BioID or APEX2 fused to ELAC2 could map its protein interaction network in different subcellular compartments, providing insights into compartment-specific functions.

Mass spectrometry-based proteomics offers another powerful approach, particularly for studying post-translational modifications of ELAC2 that may regulate its dual localization between mitochondria and nucleus . Targeted proteomics using selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) can provide absolute quantification of ELAC2 levels across tissues or disease states without antibody limitations. For functional studies, CLIP-seq (crosslinking immunoprecipitation followed by sequencing) could identify the exact RNA substrates processed by ELAC2 in vivo, extending our understanding beyond its known tRNA processing functions .

Emerging single-cell technologies might reveal cell-type-specific expression patterns and functions of ELAC2 that are obscured in bulk analyses. Additionally, the development of small molecule probes or activity-based protein profiling tools specific to ELAC2's enzymatic function could enable dynamic monitoring of its activity rather than just protein levels. These complementary approaches, integrated with traditional antibody-based methods, promise to provide a more comprehensive understanding of ELAC2's roles in normal physiology and disease states .

How might researchers integrate ELAC2 studies with broader mitochondrial biology investigation?

Integrating ELAC2 research with broader mitochondrial biology investigations offers several productive research avenues. Since ELAC2 is predominantly localized in mitochondria and functions in mitochondrial tRNA processing, researchers should examine how ELAC2 activity coordinates with the mitochondrial transcription and translation machinery. Experimental approaches could include ribosome profiling of mitochondrial translation in cells with manipulated ELAC2 levels to assess effects on protein synthesis. Additionally, researchers might explore connections between ELAC2 function and mitochondrial DNA maintenance, as altered tRNA processing could potentially impact mitochondrial gene expression and genomic stability.

Investigating the relationship between ELAC2 activity and mitochondrial stress responses could reveal how its function adapts to metabolic demands. Researchers could expose cells to various mitochondrial stressors (e.g., respiratory chain inhibitors, oxidative stress inducers) and monitor changes in ELAC2 expression, localization, and activity using validated antibodies . Mitochondrial dynamics, including fusion, fission, and mitophagy, represent another important area for integration; immunofluorescence studies could examine ELAC2's distribution during these processes using antibody dilutions of approximately 1:50-1:500 .

From a disease perspective, researchers should investigate ELAC2's role in mitochondrial disorders beyond its established connections to cancer . This could involve screening mitochondrial disease patient samples for ELAC2 variants or expression changes. Finally, considering the dual localization of ELAC2 in mitochondria and nucleus , exploring the protein's potential role in mitochondrial-nuclear communication could provide insights into how these compartments coordinate activities. Such integrative approaches would position ELAC2 research within the broader context of mitochondrial biology and potentially reveal novel functions beyond its established role in tRNA processing .

What are the most promising translational aspects of ELAC2 research for disease understanding?

The translational potential of ELAC2 research spans several disease areas, with cancer applications being particularly promising. Given ELAC2's identification as a potential prostate cancer susceptibility gene , researchers should further explore its utility as a biomarker for cancer risk stratification or prognosis. This could involve developing standardized immunohistochemical protocols using validated antibodies at dilutions of 1:50-1:500 for clinical tissue analysis. Additionally, investigating the functional consequences of ELAC2 germline variants (particularly Ser217Leu and Ala541Thr) in cellular and animal models could elucidate mechanisms underlying cancer predisposition.

Beyond cancer, ELAC2's critical role in mitochondrial tRNA processing suggests potential involvement in mitochondrial diseases. Researchers should examine ELAC2 expression and function in patients with unexplained mitochondrial dysfunction, particularly those with defects in mitochondrial protein synthesis. For neurodegenerative diseases with mitochondrial components, such as Parkinson's and Alzheimer's, investigating ELAC2's role in maintaining neuronal mitochondrial function could reveal novel pathogenic mechanisms or therapeutic targets.

From a therapeutic perspective, developing small molecule modulators of ELAC2 activity could have applications in both cancer and mitochondrial disorders. In cancer contexts where ELAC2 might be overexpressed or overactive, inhibitors could potentially slow tumor growth. Conversely, in conditions with impaired ELAC2 function, activators or stabilizers might restore normal mitochondrial RNA processing. These translational directions build upon ELAC2's established biological roles while extending into clinically relevant applications that could ultimately impact patient care .

What resources are available to researchers studying ELAC2?

Researchers investigating ELAC2 have access to a diverse array of resources spanning reagents, databases, and literature. Commercial antibodies from multiple manufacturers provide essential tools for protein detection, including Proteintech's polyclonal (10071-1-AP) and recombinant (83468-6-RR) antibodies , as well as Novus Biologicals' NBP3-12934 . These antibodies have been validated for various applications including Western blot, immunohistochemistry, immunofluorescence, flow cytometry, and immunoprecipitation, with detailed technical information available on manufacturer websites.

Genomic and proteomic databases offer valuable ELAC2 reference information, including UniProt (ID: Q9BQ52) , which provides comprehensive protein annotation, and NCBI Gene (ID: 60528) , which includes genomic and transcriptomic data. For researchers interested in genetic aspects, ELAC2's genomic location on chromosome 17p12 and reference sequence information (NC_000017.11; 12991612..13018064, complement) are available . Cell lines known to express detectable levels of ELAC2, such as DU 145, HeLa, HepG2, MCF-7, Raji, and A431, provide biological systems for functional studies .

The scientific literature contains seminal papers describing ELAC2's discovery and characterization, including Tavtigian et al. (PMID: 11175785), which identified ELAC2 as a candidate prostate cancer susceptibility gene, and Takaku et al. (PMID: 12711671), which characterized its tRNA 3' processing endoribonuclease activity . These foundational publications, along with more recent research papers, provide essential context and methodological approaches for contemporary ELAC2 investigations across basic science and disease-oriented research contexts .

How should researchers interpret contradictory findings in ELAC2 literature?

When encountering contradictory findings in ELAC2 literature, researchers should systematically evaluate several key factors that might explain the discrepancies. First, consider differences in experimental models; ELAC2 expression and function may vary between cell types, tissues, and species. While some antibodies like 10071-1-AP show cross-reactivity with human, mouse, and rat ELAC2 , others like 83468-6-RR are validated only for human samples . These species-specific differences could contribute to apparently conflicting results when comparing studies using different model systems.

Methodological variations represent another major source of potential discrepancies. Different antibodies target distinct epitopes of ELAC2 , potentially leading to variable detection efficiency depending on protein conformation, post-translational modifications, or protein-protein interactions. When comparing studies, carefully examine the specific antibody used, its dilution (ranging from 1:50-1:50000 depending on application and antibody) , and detection methods. Similarly, variations in sample preparation, such as different fixation protocols for immunohistochemistry or lysis conditions for Western blot, can significantly impact results.