GTPBP4 Antibody

What is GTPBP4 and what are its primary cellular functions?

GTPBP4 (GTP binding protein 4) is a GTPase that functions as a molecular switch, alternating between active (GTP-bound) and inactive (GDP-bound) states to regulate various cellular processes. It plays a crucial role in the biogenesis of the 60S ribosomal subunit, which is essential for protein synthesis . Additionally, GTPBP4 acts as a TP53 repressor, preventing TP53 stabilization and cell cycle arrest, thereby influencing cell proliferation and survival pathways . Research indicates that GTPBP4 interacts with multiple ribosomal and nucleolar proteins to efficiently regulate ribosome assembly, which is fundamental to cellular growth and metabolism . The protein is encoded by the GTPBP4 gene with a calculated molecular weight of 74 kDa and consists of 634 amino acids, making it an important component of cellular regulatory networks .

What are the most validated applications for GTPBP4 antibodies in research?

GTPBP4 antibodies have been validated for multiple research applications, with Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF/ICC), Immunoprecipitation (IP), and ELISA being the most common and reliable methods . Western blotting remains the most widely used technique, with recommended dilutions typically ranging from 1:2000 to 1:8000, allowing for sensitive detection of GTPBP4 protein expression in various cell and tissue lysates . Immunofluorescence applications typically employ dilutions between 1:200 and 1:800, enabling researchers to visualize the subcellular localization of GTPBP4, which has been observed in both the cytoplasm and nucleus of cancer cells . According to Antibodypedia data, several antibodies have been extensively validated with multiple references, including Proteintech's 13897-1-AP, which has been cited in 5 publications and validated for WB, ELISA, ICC, IP, and IHC applications .

How should researchers select the appropriate GTPBP4 antibody for their experiments?

Researchers should base their selection on several critical factors, including the specific application, target species, and validation status of the antibody. First, confirm the antibody's reactivity with your species of interest, as validated options are available for human, mouse, and sometimes rat samples . Second, evaluate the validation data for your intended application—the most extensively validated GTPBP4 antibodies demonstrate consistent performance across Western blot, immunohistochemistry, and immunofluorescence applications . Third, consider the clonality of the antibody; polyclonal antibodies like 13897-1-AP offer high sensitivity across multiple epitopes, while monoclonal antibodies like ab92342 [EPR3500] provide higher specificity for particular epitopes . Finally, review published research that has successfully employed the antibody in similar experimental conditions to your planned studies, focusing on those with peer-reviewed validation data rather than relying solely on manufacturer claims .

What are the optimal storage and handling conditions for GTPBP4 antibodies?

GTPBP4 antibodies require specific storage and handling conditions to maintain their functionality and specificity. Most GTPBP4 antibodies should be stored at -20°C, where they typically remain stable for at least one year after shipment . The standard storage buffer for these antibodies generally contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps prevent degradation and preserve antibody activity . For smaller aliquots (20μl), some products contain 0.1% BSA as a stabilizer, though aliquoting is generally considered unnecessary for -20°C storage . When working with these antibodies, it's advisable to avoid repeated freeze-thaw cycles, which can compromise antibody integrity and performance. During experimental procedures, researchers should closely follow manufacturer-recommended dilutions, which vary by application (WB: 1:2000-1:8000; IF/ICC: 1:200-1:800; IP: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate), as improper dilution can lead to suboptimal results or background issues .

How can GTPBP4 antibodies be used to study cancer progression mechanisms?

GTPBP4 antibodies serve as crucial tools for investigating cancer progression mechanisms due to GTPBP4's emerging role as a potential oncogene in several malignancies. In lung adenocarcinoma research, these antibodies have been instrumental in demonstrating that GTPBP4 is significantly upregulated in tumor tissues compared to adjacent normal tissues, with immunohistochemical staining revealing high expression in both cytoplasmic and nuclear compartments of cancer cells . Western blot analyses using GTPBP4 antibodies have enabled researchers to quantify elevated expression in various lung cancer cell lines (H838, H2347, Calu-1, A549) compared to normal lung cells, providing critical insights into cancer-specific protein expression patterns . Furthermore, GTPBP4 antibodies have been essential in validating knockdown or knockout models, where reduced GTPBP4 expression correlates with inhibited cell proliferation and invasion in both in vitro cell studies and in vivo mouse models . These applications extend to liver cancer research, where antibody-based detection has revealed that GTPBP4 overexpression correlates with increased tumor growth, while its downregulation inhibits cancer cell proliferation, suggesting it could serve as both a prognostic biomarker and therapeutic target .

What technical considerations are important when using GTPBP4 antibodies for Western blotting?

When using GTPBP4 antibodies for Western blotting, researchers should consider several technical aspects to ensure reliable and reproducible results. First, sample preparation is critical—validated Western blot protocols have successfully detected GTPBP4 in various sample types including HeLa cells, mouse testis tissue, human kidney tissue, and HepG2 cells . Second, researchers should be aware of the expected molecular weight; while the calculated molecular weight of GTPBP4 is 74 kDa, some antibodies detect a band around 80 kDa, indicating possible post-translational modifications or isoform detection . Third, gel percentage selection is important—7.5% SDS-PAGE has been successfully used for GTPBP4 detection, providing appropriate resolution for this molecular weight range . Fourth, proper antibody dilution is essential, with effective dilutions ranging from 1:500 to 1:8000 depending on the specific antibody and sample type . Finally, researchers should optimize blocking conditions and consider validation controls—the use of GTPBP4 knockdown or knockout samples as negative controls can confirm antibody specificity and minimize interpretation errors in complex experimental settings .

How can researchers effectively use GTPBP4 antibodies in immunofluorescence and immunohistochemistry applications?

For effective immunofluorescence and immunohistochemistry applications using GTPBP4 antibodies, researchers should implement specific optimization strategies. When performing immunofluorescence, dilutions of 1:200-1:800 have proven effective for detecting GTPBP4 in various cell types, with MDCK cells specifically validated for positive detection . Fixation methods significantly impact results—while many protocols use paraformaldehyde fixation, researchers should validate optimal fixation conditions for their specific cell type to preserve GTPBP4 epitopes. For immunohistochemistry on paraffin-embedded tissues, antibodies like ab92342 at 1:250 dilution have successfully detected GTPBP4 in human kidney tissues, with recommendations to use HRP/AP polymerized antibodies as secondary detection reagents . Importantly, subcellular localization analysis requires careful optimization—published studies have shown GTPBP4 expression in both cytoplasm and nucleus of lung cancer cells, requiring high-resolution imaging and appropriate controls to distinguish specific staining patterns . When interpreting results, researchers should consider that GTPBP4 expression varies significantly between cancer and normal tissues, making parallel staining of control tissues essential for accurate assessment of expression differences .

What strategies should be employed for validating GTPBP4 antibody specificity in experimental models?

Comprehensive validation of GTPBP4 antibody specificity requires multiple complementary approaches to ensure experimental rigor. The gold standard approach involves genetic validation through GTPBP4 knockdown or knockout models—several studies have successfully implemented GTPBP4 shRNA in A549 and Calu-1 cells or GTPBP4 knockout in LLC cells, creating essential negative controls for antibody specificity testing . Western blot analysis should demonstrate appropriate molecular weight detection (approximately 74 kDa) across multiple cell lines and tissue samples, as validated with antibodies like 13897-1-AP in HeLa cells, mouse testis tissue, human kidney tissue, and HepG2 cells . Cross-validation with multiple antibodies targeting different GTPBP4 epitopes provides additional confidence—comparing results from polyclonal antibodies like 13897-1-AP with monoclonal antibodies such as ab92342 [EPR3500] can confirm detection consistency . Immunoprecipitation followed by mass spectrometry can further verify antibody-antigen interaction specificity, particularly valuable for complex experimental systems. Finally, tissue expression pattern analysis should align with known GTPBP4 distribution—published data demonstrates elevated expression in various cancer tissues compared to normal counterparts, providing a reference pattern for validation .

What experimental approaches are effective for studying GTPBP4 function in cancer models using GTPBP4 antibodies?

Investigating GTPBP4 function in cancer models requires integrated experimental approaches centered around antibody-based detection methods. For in vitro functional studies, researchers have successfully employed RNA interference techniques (shRNA) to knockdown GTPBP4 in lung cancer cell lines (A549 and Calu-1), with antibody-based Western blot verification confirming successful expression reduction . Cell proliferation assays (CCK-8) combined with GTPBP4 antibody-based expression analysis have revealed that GTPBP4 knockdown significantly decreases cancer cell proliferation and invasion capabilities in transwell assays . In vivo models utilizing GTPBP4 knockout in mouse lung cancer cells (LLC-GTPBP4 KO) have demonstrated reduced lung weight and decreased surface nodules compared to wild-type controls, with immunohistochemical verification using GTPBP4 antibodies confirming expression differences in tumor tissues . Molecular mechanism investigations suggest GTPBP4 may promote cancer progression through epithelial-mesenchymal transition (EMT) regulation, where antibody-based detection of EMT markers alongside GTPBP4 can elucidate pathway interactions . Additionally, co-immunoprecipitation experiments using GTPBP4 antibodies have helped identify interaction partners such as NIFK, WDR12, and RPF2, contributing to understanding of GTPBP4's role in rRNA processing and GTP binding within cancer contexts .

How can researchers troubleshoot inconsistent results when using GTPBP4 antibodies in cancer tissue samples?

When encountering inconsistent results with GTPBP4 antibodies in cancer tissue samples, researchers should implement a systematic troubleshooting approach addressing multiple variables. First, tissue preparation variations can significantly impact antibody performance—researchers should standardize fixation protocols (duration, fixative composition) and antigen retrieval methods, as GTPBP4 epitopes may be differentially affected by processing methods . Second, antibody selection is critical—inconsistencies may arise from using antibodies targeting different epitopes, so researchers should compare results using both polyclonal antibodies (e.g., 13897-1-AP) that recognize multiple epitopes and monoclonal antibodies (e.g., ab92342) with higher epitope specificity . Third, expression heterogeneity within tumors must be considered—GTPBP4 expression can vary between tumor regions and cell types, requiring multiple sampling areas and comprehensive image analysis to accurately quantify expression patterns . Fourth, careful controls must be implemented—using both positive controls (tissues with known high GTPBP4 expression such as HeLa cells, testis tissue) and negative controls (GTPBP4 knockdown tissues) to validate staining specificity . Finally, correlation with other detection methods including RNA expression analysis (qPCR) can help validate protein-level findings and resolve discrepancies between antibody-based detection results .

How can GTPBP4 antibodies contribute to developing potential cancer therapeutics and biomarkers?

GTPBP4 antibodies are instrumental in advancing cancer therapeutic and biomarker development through multiple research avenues. Target validation studies utilizing these antibodies have demonstrated that GTPBP4 is significantly upregulated in lung adenocarcinoma and liver cancer tissues compared to normal controls, establishing it as a promising therapeutic target . Diagnostic biomarker development is supported by antibody-based tissue analysis showing that GTPBP4 has excellent diagnostic value for lung cancer with an area under the ROC curve of 0.882, suggesting potential utility in early cancer detection . Therapeutic response monitoring applications are emerging as knockdown studies using GTPBP4 antibodies for validation have shown that reducing GTPBP4 expression inhibits cancer cell proliferation and invasion, providing a measurable marker for intervention efficacy . Drug development pipelines can leverage GTPBP4 antibodies for high-throughput screening of compounds that modulate GTPBP4 expression or function, with subsequent validation of hit compounds using antibody-based detection methods. Furthermore, companion diagnostic development could pair GTPBP4 antibody-based tissue analysis with targeted therapies, potentially creating precision medicine approaches for patients with tumors showing high GTPBP4 expression, which correlates with poorer prognosis in liver cancer patients .

What are the current limitations of available GTPBP4 antibodies and how might they be addressed in future research?

Current GTPBP4 antibodies face several limitations that require methodological advances to overcome in future research. Epitope coverage presents a significant challenge—many available antibodies target limited regions of the 634 amino acid GTPBP4 protein, potentially missing important functional domains or post-translational modifications that may be relevant in disease contexts . Cross-reactivity concerns arise particularly with polyclonal antibodies, which may detect closely related GTP-binding proteins; future development of highly specific monoclonal antibodies with thoroughly validated specificity would address this limitation . Isoform detection remains problematic—current antibodies may not distinguish between potential GTPBP4 isoforms, as suggested by the detection of bands at both 74 kDa (calculated) and 80 kDa (observed) in Western blots, requiring isoform-specific antibodies for comprehensive analysis . Application versatility varies significantly between antibodies, with many validated for limited applications; development of antibodies that perform consistently across multiple techniques (WB, IHC, IF, IP, ChIP) would enhance experimental flexibility . Finally, quantitative limitations exist as most current antibodies are optimized for qualitative rather than quantitative analysis; development of standardized protocols using calibrated recombinant GTPBP4 standards could enable more precise quantification of expression levels across different experimental systems .

How might single-cell analysis techniques incorporate GTPBP4 antibodies to advance cancer research?

Single-cell analysis techniques incorporating GTPBP4 antibodies present transformative opportunities for advancing cancer research through comprehensive cellular heterogeneity assessment. Mass cytometry (CyTOF) could integrate metal-conjugated GTPBP4 antibodies into multi-parameter panels, enabling simultaneous detection of GTPBP4 alongside dozens of other cancer-relevant proteins and signaling molecules in individual cells, revealing co-expression patterns impossible to detect in bulk analysis . Single-cell immunofluorescence techniques utilizing validated GTPBP4 antibodies (dilutions 1:200-1:800) could map subcellular localization patterns at unprecedented resolution, potentially revealing functional differences between cytoplasmic and nuclear GTPBP4 pools observed in cancer cells . Microfluidic approaches could combine GTPBP4 antibody-based sorting with single-cell transcriptomics, allowing researchers to correlate GTPBP4 protein levels with genome-wide expression profiles in the same cells, potentially identifying novel mechanistic relationships. Spatial transcriptomics/proteomics methodologies incorporating GTPBP4 antibodies could map expression patterns within the tumor microenvironment, revealing interactions between GTPBP4-expressing cancer cells and surrounding stromal components that might influence cancer progression. These advanced techniques would significantly extend current knowledge derived from bulk analyses, where GTPBP4 overexpression has been associated with cancer progression and poorer outcomes in lung and liver cancers .

What are the recommended antibody dilutions and experimental conditions for different GTPBP4 detection methods?

Optimal detection of GTPBP4 requires application-specific antibody dilutions and carefully controlled experimental conditions to ensure reliable results. For Western blotting applications, recommended dilutions vary between antibodies but typically range from 1:500 to 1:8000, with Proteintech's 13897-1-AP validated at 1:2000-1:8000 and Abcam's ab184124 effective at 1:500 dilution . Immunoprecipitation protocols should utilize 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate, with successful results demonstrated in HeLa cells and mouse testis tissue samples . Immunofluorescence and immunocytochemistry applications require dilutions of 1:200-1:800, with positive detection validated in MDCK cells and various cancer cell lines . Immunohistochemistry on paraffin-embedded tissues has been successful with dilutions around 1:250, as demonstrated with ab92342 in human kidney tissue sections, preferably using HRP/AP polymerized antibodies as secondary detection reagents . Across all applications, researchers should note that optimal results may require method-specific sample preparation—successful Western blotting has been demonstrated with 7.5% SDS-PAGE gels, while immunohistochemistry requires appropriate antigen retrieval methods to expose GTPBP4 epitopes that may be masked during fixation .

What positive and negative controls are recommended when working with GTPBP4 antibodies?

Implementing appropriate controls is essential for accurate interpretation of GTPBP4 antibody experiments across different applications. For positive controls in Western blotting, researchers should consider using validated sample types including HeLa cells, HepG2 cells, mouse testis tissue, and human kidney tissue, which have been confirmed to express detectable levels of GTPBP4 . Immunofluorescence and immunocytochemistry experiments can utilize MDCK cells as positive controls, while human kidney tissue has been validated for immunohistochemistry applications . Negative controls should ideally include GTPBP4 knockdown or knockout models, which have been successfully generated in lung cancer cell lines (A549, Calu-1) and mouse models (LLC-GTPBP4 KO), providing essential specificity verification for antibody-based detection methods . Technical negative controls should include primary antibody omission and isotype controls (rabbit IgG for most GTPBP4 antibodies), helping distinguish specific signal from background or non-specific binding . For validation of novel applications or sample types, researchers should implement expression correlation controls—comparing protein detection using antibodies to mRNA expression using qPCR or RNA-seq data, which has successfully validated GTPBP4 expression patterns in cancer tissue studies .

What are the comparative performance metrics of different GTPBP4 antibodies across applications?

This comprehensive comparison table synthesizes data from multiple sources to provide researchers with performance metrics across different GTPBP4 antibodies . The Proteintech 13897-1-AP antibody demonstrates the most extensive validation with five publications and verification across multiple applications and sample types, making it particularly suitable for researchers requiring a versatile reagent . The Abcam monoclonal antibody ab92342 (EPR3500) offers the advantage of high specificity typical of monoclonal antibodies but has been validated in fewer applications, primarily Western blot and immunohistochemistry . When selecting an antibody for novel applications or sample types, researchers should consider that polyclonal antibodies generally offer broader epitope recognition but potentially increased background, while monoclonal antibodies provide greater specificity but may be more sensitive to epitope masking during sample processing .

What experimental protocols have been validated for studying GTPBP4 in cancer models?

This table outlines validated experimental protocols for studying GTPBP4 in cancer models, providing researchers with methodological frameworks that have yielded significant insights into GTPBP4's role in tumor progression . The integration of in vitro knockdown/knockout approaches with in vivo tumor models has established a causal relationship between GTPBP4 expression and cancer progression, with antibody-based detection methods central to verifying expression changes . Tissue microarray analysis has revealed consistent upregulation of GTPBP4 in cancer tissues compared to normal controls, highlighting its potential as a biomarker . Emerging mechanistic studies suggest GTPBP4's oncogenic effects may operate through epithelial-mesenchymal transition pathways, providing direction for future therapeutic targeting strategies . Researchers designing new studies should consider this multi-level approach, combining cellular and animal models with comprehensive expression analysis in patient samples to establish clinically relevant insights into GTPBP4 function .

What performance metrics should researchers consider when validating a new GTPBP4 antibody?

This comprehensive validation framework provides researchers with structured performance metrics for evaluating new GTPBP4 antibodies, ensuring experimental reliability before implementing them in critical research applications . Specificity testing is particularly important given the presence of multiple GTP-binding proteins in cells, with knockout or knockdown models serving as the gold standard for validation . Sensitivity assessment helps researchers determine optimal working concentrations, with established GTPBP4 antibodies demonstrating effective performance at dilutions ranging from 1:200 to 1:8000 depending on the application . Subcellular localization validation is especially relevant for GTPBP4, which has been observed in both cytoplasmic and nuclear compartments in cancer cells, requiring careful confocal microscopy analysis with appropriate markers . Researchers should document comprehensive validation data for new antibodies, as this transparency enhances research reproducibility and facilitates appropriate method selection for specific experimental questions .