AGP26 Antibody

What is the AGP26.10 antibody and what target does it recognize?

AGP26.10 is a monoclonal mouse IgG1 antibody with kappa light chain that specifically recognizes the integral membrane pore glycoprotein gp210, a component of the nuclear matrix-pore complex-lamina (NMPCL) in Drosophila . This antibody was generated using purified native NMPCL glycoprotein as an immunogen, and it binds to the full-length protein with a molecular weight of approximately 209 kDa . The antibody was deposited to the Developmental Studies Hybridoma Bank (DSHB) by P.A. Fisher from SUNY at Stony Brook's Department of Pharmacological Sciences and has been assigned the Research Resource Identifier (RRID) AB_528271 . Understanding the molecular target of this antibody is essential for researchers designing experiments to study nuclear pore complex structure and function, particularly in Drosophila models where it has confirmed species reactivity . The specific epitope location has not been mapped, which should be considered when planning experiments where epitope accessibility might be affected by experimental conditions or protein conformational changes .

What are the recommended applications for AGP26.10 antibody?

The AGP26.10 antibody has been validated for two primary applications: immunofluorescence microscopy and Western blot analysis . For immunofluorescence applications, the antibody can effectively visualize the nuclear envelope in fixed Drosophila cells, showing the characteristic punctate pattern of nuclear pore complexes around the nuclear periphery . In Western blot applications, the antibody recognizes the 209 kDa gp210 protein under appropriate denaturing conditions, making it valuable for protein expression studies and biochemical analyses . While these are the established applications, researchers often adapt antibodies for additional techniques such as immunoprecipitation or chromatin immunoprecipitation, though such extended applications would require validation by the individual researcher . The methodological approach for each application should include appropriate positive and negative controls, especially when working with tissues or cell types not previously tested with this antibody . Researchers should also consider optimization of antibody concentration for each specific application to achieve the best signal-to-noise ratio in their experimental system .

How should AGP26.10 antibody be stored and handled for optimal performance?

For optimal antibody performance and longevity, AGP26.10 requires specific storage and handling protocols that preserve its immunoreactivity . While many cell products can be maintained at 4°C for extended periods, shelf-life at this temperature is highly variable; therefore, for immediate use, short-term storage at 4°C for up to two weeks is recommended . For long-term storage, the antibody solution should be divided into small aliquots of no less than 20 μl to minimize freeze-thaw cycles, which can degrade antibody activity, and stored at either -20°C or -80°C . When working with concentrate or bioreactor products, adding an equal volume of glycerol as a cryoprotectant may enhance stability during freezing . Prior to use, antibody aliquots should be thawed gradually on ice to maintain protein integrity, and centrifuged briefly to collect all liquid at the bottom of the tube . Researchers should avoid repeated freeze-thaw cycles and always handle the antibody using sterile technique to prevent microbial contamination that could compromise experimental results . It's worth noting that the antibody contains the antimicrobial ProClin, which enhances its shelf stability but should be considered when designing experiments where antimicrobial agents might interfere .

What considerations should be made for species cross-reactivity with AGP26.10?

The AGP26.10 antibody has confirmed species reactivity with Drosophila, as it was raised against the Drosophila nuclear pore complex glycoprotein gp210 . When planning experiments with this antibody, researchers should be aware that its specificity for other species has not been documented in the provided information, which could limit its application in comparative studies across different model organisms . Nuclear pore complex proteins often show evolutionary conservation, so there may be potential cross-reactivity with homologous proteins in closely related species, though this would require empirical validation through careful control experiments . For researchers interested in exploring cross-reactivity, preliminary experiments should include positive controls using Drosophila samples alongside test samples from the species of interest, with identical processing conditions to allow direct comparison of binding specificity and signal intensity . Western blot analysis can serve as an initial screen for cross-reactivity by revealing whether the antibody recognizes proteins of the expected molecular weight in non-Drosophila samples . If cross-reactivity is observed, sequence alignment of the target protein across species can provide insight into the degree of conservation in potential epitope regions, enhancing interpretation of experimental results .

What methodological approaches can optimize immunofluorescence experiments with AGP26.10?

To achieve optimal immunofluorescence results with AGP26.10 antibody, researchers should implement a systematic optimization approach that addresses multiple experimental variables . First, fixation methods significantly impact epitope preservation and accessibility; while paraformaldehyde fixation (4%, 10-15 minutes) often provides a good starting point for nuclear envelope proteins, comparing multiple fixation protocols (including methanol or glutaraldehyde-based methods) can identify conditions that best preserve the gp210 epitope recognized by AGP26.10 . Second, permeabilization conditions should be carefully titrated, as nuclear pore complex proteins may require stronger permeabilization (e.g., 0.5% Triton X-100) than typical cytoplasmic proteins to allow antibody access to the nuclear envelope . Third, blocking conditions should be optimized by testing different blocking agents (BSA, normal serum, commercial blocking solutions) at various concentrations and incubation times to minimize background while preserving specific signal . Fourth, antibody dilution series (typically starting from 1:100 to 1:1000) should be tested to determine the optimal concentration that maximizes specific signal while minimizing background . Finally, detection systems should be evaluated, comparing direct fluorophore-conjugated secondary antibodies with signal amplification methods (such as biotin-streptavidin systems) particularly for tissues where target expression might be low or in multi-labeling experiments where signal separation is critical .

What are the critical parameters for successful Western blot detection using AGP26.10?

Successfully detecting gp210 by Western blot using AGP26.10 requires careful attention to several critical parameters due to the high molecular weight (209 kDa) of the target protein . First, sample preparation must preserve protein integrity while ensuring complete denaturation; this typically requires a buffer containing SDS (1-2%), reducing agents (DTT or β-mercaptoethanol), and protease inhibitors, with moderate heating (70°C for 10 minutes rather than boiling) to prevent aggregation of large transmembrane proteins . Second, gel electrophoresis should utilize low percentage gels (6-8% acrylamide) or gradient gels (4-15%) to allow proper separation and resolution of high molecular weight proteins, with extended running times at lower voltage to prevent band distortion . Third, transfer conditions must be optimized for large proteins, typically using wet transfer systems with methanol-free transfer buffer containing 0.05-0.1% SDS to facilitate movement of large proteins from gel to membrane, with extended transfer times (overnight at low amperage) or specialized systems designed for high molecular weight proteins . Fourth, membrane blocking and antibody incubation should be performed with optimized blocking agents (typically 5% non-fat dry milk or BSA) and antibody dilutions determined empirically through titration experiments, with extended primary antibody incubation (overnight at 4°C) to ensure sufficient binding . Finally, appropriate controls should be included in each experiment, such as positive controls from Drosophila cells/tissues known to express gp210, negative controls from tissues where expression is absent, and molecular weight markers that extend into the high molecular weight range .

How can researchers validate AGP26.10 specificity for novel applications?

Validating AGP26.10 antibody specificity for novel applications requires a multi-faceted approach to establish confidence in experimental results . The first validation step should include performing parallel experiments with multiple antibodies targeting different epitopes of the same protein (gp210) if available, which can confirm that the observed signals truly represent the target protein rather than non-specific binding . Second, researchers should conduct knockdown or knockout experiments using RNAi or CRISPR-Cas9 to reduce or eliminate gp210 expression, followed by immunostaining or Western blotting with AGP26.10 to confirm signal reduction proportional to the decreased target expression . Third, heterologous expression systems can be employed where gp210 is overexpressed in cells that normally express low levels of the protein, with subsequent detection using AGP26.10 to verify signal increase corresponding to increased target abundance . Fourth, peptide competition assays can be performed where the antibody is pre-incubated with purified gp210 protein or peptides containing the presumed epitope sequence before application to samples, which should result in signal ablation if the antibody is specific . Finally, for novel applications such as immunoprecipitation or chromatin immunoprecipitation, mass spectrometry analysis of the immunoprecipitated material can provide definitive identification of the proteins recognized by AGP26.10, confirming specificity and potentially revealing previously unknown interacting partners .

What approaches can address background and non-specific binding issues with AGP26.10?

Non-specific binding and background signals can significantly complicate data interpretation when using AGP26.10 antibody, particularly in complex tissues or when applying the antibody to new experimental systems . To address these challenges, researchers should first optimize antibody concentration through careful titration experiments, as excessive antibody can lead to increased non-specific binding while insufficient antibody may result in weak specific signals that are difficult to distinguish from background . Second, blocking protocols should be enhanced by testing different blocking agents (BSA, casein, commercial blockers, normal serum matched to the secondary antibody species) at various concentrations and incubation times to identify the most effective combination for reducing background while preserving specific signal . Third, washing steps should be optimized by increasing the number, duration, and stringency of washes (e.g., higher salt concentration or addition of mild detergents like Tween-20) to remove weakly bound antibody without affecting specific interactions . Fourth, pre-adsorption of the antibody with tissues or cell lysates from species or samples that don't express the target can help remove antibodies that bind to conserved epitopes or common cellular components . Finally, secondary antibody controls (samples processed with secondary antibody only, omitting primary antibody) and isotype controls (using non-specific mouse IgG1 at the same concentration as AGP26.10) should be included in all experiments to distinguish between primary antibody-specific background and issues related to the detection system or sample preparation .

How can AGP26.10 be used to study nuclear pore complex dynamics in developmental contexts?

The AGP26.10 antibody presents a valuable tool for investigating nuclear pore complex (NPC) dynamics throughout Drosophila development due to its specific recognition of the integral membrane pore glycoprotein gp210 . To effectively study developmental changes in NPC composition and distribution, researchers can implement time-course experiments collecting samples at defined developmental stages from embryo to adult, processing all samples in parallel with standardized immunostaining protocols to enable direct comparison of gp210 expression patterns and levels . Combining AGP26.10 with antibodies against other NPC components (such as nucleoporins or lamins) in multi-color immunofluorescence experiments can reveal the temporal sequence of NPC assembly during nuclear envelope formation and maturation, particularly during rapid embryonic divisions where nuclear envelope dynamics are heightened . For quantitative analysis, automated image analysis pipelines can be developed to measure parameters such as NPC density, clustering, and signal intensity across developmental timepoints, providing objective metrics of NPC remodeling . Live imaging experiments in developing tissues can be designed using fluorescently-tagged secondary antibodies against AGP26.10 introduced via microinjection of early embryos, though such approaches require careful validation to ensure antibody binding doesn't disrupt normal NPC function . For tissue-specific analysis, AGP26.10 can be used in conjunction with tissue-specific markers to compare NPC characteristics across different cell types during development, potentially revealing tissue-specific requirements for nuclear transport during differentiation .

What are the potential applications of AGP26.10 in studying neurological disorders?

While AGP26.10 specifically targets the Drosophila nuclear pore complex protein gp210, research on nuclear pore complexes has broader implications for understanding neurological disorders, especially those involving autoimmune mechanisms . Recent studies have identified autoantibodies against nuclear pore complex proteins like ARHGAP26 (RhoGTPase-activating protein 26) in patients with neurological conditions including autoimmune cerebellar ataxia, cognitive impairment, and affective disorders . Although AGP26.10 and anti-ARHGAP26 target different proteins, researchers can develop parallel experimental approaches using these antibodies to investigate the structural integrity and function of nuclear pore complexes in neuronal cells . In Drosophila models of neurodegeneration, AGP26.10 can be employed to examine changes in nuclear envelope architecture and nucleocytoplasmic transport that might precede or accompany neuronal dysfunction . Comparative studies between the nuclear pore complex alterations observed in Drosophila models (detected with AGP26.10) and those seen in human patients with anti-nuclear pore complex autoantibodies could reveal conserved mechanisms of nuclear pore complex dysfunction in neurological disease . Additionally, the methodological approaches developed for AGP26.10 in Drosophila systems can inform experimental design for studying autoantibodies against nuclear pore complex components in human samples, potentially advancing understanding of the pathogenesis of autoimmune neurological disorders .

How does sample preparation affect AGP26.10 antibody performance?

Sample preparation significantly impacts AGP26.10 antibody performance across different experimental applications, requiring careful optimization for reliable results . For immunofluorescence applications, fixation method is critical—aldehyde-based fixatives (particularly 4% paraformaldehyde) generally preserve epitope structure while maintaining cellular architecture, though fixation time should be empirically determined as over-fixation can mask epitopes within the nuclear pore complex . Permeabilization conditions directly affect antibody accessibility to nuclear envelope components; for transmembrane proteins like gp210, stronger permeabilization with detergents such as 0.5% Triton X-100 or NP-40 may be necessary compared to protocols for cytoplasmic proteins . For Western blot applications, particular attention must be paid to extraction conditions for this high molecular weight, membrane-integrated protein; sample buffers should contain sufficient detergent (1-2% SDS) and reducing agents, with extraction temperatures carefully controlled to solubilize membrane proteins without causing aggregation . Tissue preservation methods also influence antibody performance—fresh or flash-frozen tissues typically yield better results than formalin-fixed paraffin-embedded samples, which may require antigen retrieval methods to restore epitope recognition . For Drosophila developmental studies, the cuticle and peritrophic membrane can create penetration barriers for antibodies, necessitating extended permeabilization steps or physical disruption techniques to ensure antibody access to internal tissues . Standardizing sample preparation protocols is essential for reproducibility, particularly when comparing gp210 expression or localization across different experimental conditions or genetic backgrounds .

What controls should be included when using AGP26.10 in experimental protocols?

Rigorous control experiments are essential when working with AGP26.10 antibody to ensure data validity and facilitate accurate interpretation of results . Positive controls should include wild-type Drosophila samples known to express gp210, processed in parallel with experimental samples to confirm antibody functionality and establish expected signal patterns . Negative controls should incorporate samples lacking gp210 expression, such as tissues from gp210 knockout or knockdown Drosophila lines, which should show significant signal reduction proportional to the degree of protein depletion . Antibody specificity controls should include competitive inhibition experiments where AGP26.10 is pre-incubated with purified gp210 protein or peptides containing the presumed epitope before application to samples, which should abolish specific signal while leaving non-specific background unchanged . Technical controls should address secondary antibody specificity (omitting primary antibody), endogenous peroxidase activity (for HRP-based detection systems), and autofluorescence (sample processing without any antibodies) to distinguish true signals from technical artifacts . For quantitative applications, standard curve controls using recombinant gp210 protein at known concentrations can establish the linear detection range of the assay . When performing co-localization studies, single-label controls are essential to ensure spectral separation between fluorophores and absence of bleed-through, particularly important when studying nuclear pore complex proteins that form dense clusters at the nuclear envelope .

Can AGP26.10 be used in combination with other antibodies in multi-label experiments?

Multi-label experiments combining AGP26.10 with antibodies against other cellular components can provide valuable insights into nuclear pore complex biology and relationships with other cellular structures . When designing such experiments, researchers should first consider the host species of all antibodies to avoid cross-reactivity between secondary detection reagents; as AGP26.10 is a mouse monoclonal IgG1, it should ideally be paired with primary antibodies from different species (rabbit, goat, chicken, etc.) or with mouse antibodies of different isotypes that can be distinguished using isotype-specific secondary antibodies . Sequential staining protocols may be necessary when combining multiple mouse monoclonal antibodies, with complete blocking steps between each primary-secondary antibody pair to prevent cross-detection . Optimization of antibody dilutions is particularly important in multi-label experiments, as conditions optimal for single-label detection may require adjustment when antibodies are combined due to potential interactions between detection systems . Fluorophore selection should account for spectral overlap and the relative abundance of each target, assigning brighter fluorophores to less abundant proteins and ensuring sufficient spectral separation to allow clear discrimination between signals . Controls for multi-label experiments should include single-label samples for each antibody to establish baseline signal patterns and intensities, as well as fluorescence minus one (FMO) controls to identify any spectral overlap or unexpected interactions between detection systems . Advanced imaging techniques such as spectral unmixing or linear unmixing may be necessary to separate overlapping signals in densely labeled structures like the nuclear envelope .

What are the methodological considerations for using AGP26.10 in electron microscopy studies?

Adapting AGP26.10 for electron microscopy (EM) applications requires specific methodological considerations to preserve both antigenicity and ultrastructural details of nuclear pore complexes . For pre-embedding immunogold labeling, mild fixation conditions (0.5-2% paraformaldehyde with or without low concentrations of glutaraldehyde) should be used to maintain epitope recognition while providing sufficient structural preservation . Permeabilization must be carefully balanced to allow antibody access to the nuclear envelope without destroying membrane architecture; graded ethanol series or low concentrations of saponin may provide better results than stronger detergents like Triton X-100 for EM applications . For post-embedding techniques, embedding media selection is critical—acrylic resins like LR White or Lowicryl typically preserve antigenicity better than epoxy resins, though they may provide less structural detail . Immunogold particle size should be selected based on the research question: smaller gold particles (5-10 nm) offer higher spatial resolution but lower visibility, while larger particles (15-20 nm) are more visible but may obscure fine structural details . For double-labeling EM studies, AGP26.10 can be combined with antibodies against other nuclear pore complex components using gold particles of different sizes, allowing spatial relationships to be determined at ultrastructural resolution . Correlative light and electron microscopy (CLEM) approaches can be particularly valuable, using AGP26.10 for fluorescence localization to identify regions of interest followed by EM analysis of the same structures, providing both contextual information and ultrastructural details . Quantitative analysis of immunogold labeling patterns can reveal the precise distribution of gp210 within the nuclear pore complex architecture, contributing to structural models of these complex macromolecular assemblies .

How does gp210 expression detected by AGP26.10 compare across different developmental stages?

The nuclear pore complex protein gp210 recognized by AGP26.10 exhibits dynamic expression patterns throughout Drosophila development, with implications for nuclear envelope assembly and function during cellular differentiation . To comprehensively analyze these developmental changes, researchers should implement quantitative immunofluorescence or Western blot protocols using AGP26.10 across all developmental stages from embryogenesis through larval instars, pupation, and adult tissues . Early embryonic stages typically show rapid nuclear divisions with dynamic nuclear envelope breakdown and reformation, where gp210 incorporation into reassembling nuclear pore complexes can be visualized using carefully timed fixation and AGP26.10 immunostaining . Quantitative image analysis of AGP26.10 staining intensity, nuclear pore complex density, and distribution patterns can reveal stage-specific changes in nuclear envelope organization, particularly during transitions between developmental stages that involve significant tissue remodeling . Western blot analysis using AGP26.10 can complement imaging approaches by providing quantitative data on total gp210 protein levels normalized to appropriate loading controls, potentially revealing stage-specific regulation of gp210 expression . Correlation between gp210 expression patterns and developmental processes such as cell differentiation, migration, or programmed cell death can provide insights into the role of nuclear pore complex composition in regulating gene expression during development . Comparative analysis between different tissues at the same developmental stage can further reveal tissue-specific requirements for nuclear pore complex components, potentially relating to differences in transcriptional activity or nuclear transport requirements .

How might AGP26.10 contribute to understanding the relationship between nuclear pore complexes and neurological disorders?

The AGP26.10 antibody could play a significant role in elucidating connections between nuclear pore complex dysfunction and neurological disorders through several research avenues . While AGP26.10 specifically targets Drosophila gp210, the conserved nature of nuclear pore complex structures makes Drosophila an excellent model system for studying fundamental mechanisms that may apply to human disease contexts . Research has identified autoantibodies against nuclear pore complex components, including ARHGAP26, in patients with neurological disorders such as cerebellar ataxia and cognitive impairment, suggesting that nuclear pore complex dysfunction may contribute to pathogenesis . Using AGP26.10 in Drosophila models of neurodegeneration could reveal how alterations in nuclear pore complex composition affect neuronal function, providing mechanistic insights potentially relevant to human disorders . Comparative studies between nuclear pore complex alterations observed with AGP26.10 in Drosophila neurons and those seen in human neurological disorders could identify conserved pathways of nuclear pore complex dysfunction . Time-course experiments tracking nuclear pore complex changes using AGP26.10 in genetic models of neurodegeneration might reveal whether nuclear pore complex disruption precedes or follows other cellular pathologies, helping establish causative relationships rather than merely correlative observations . Furthermore, AGP26.10 could be used to evaluate potential therapeutic approaches targeting nuclear pore complex integrity or function in Drosophila models before translation to mammalian systems, potentially accelerating drug discovery for neurological disorders with nuclear pore complex involvement .

What emerging techniques could enhance the research applications of AGP26.10 antibody?

Emerging technologies present exciting opportunities to expand the research applications of AGP26.10 antibody beyond conventional immunofluorescence and Western blot analyses . Super-resolution microscopy techniques (such as STORM, PALM, or STED) can overcome the diffraction limit of conventional microscopy, potentially allowing visualization of individual nuclear pore complexes and precise localization of gp210 within the nuclear pore complex architecture when using AGP26.10 . Single-molecule tracking approaches could employ fluorescently-labeled AGP26.10 Fab fragments to study the dynamics of gp210 molecules within the nuclear envelope of living cells, providing insights into nuclear pore complex assembly and turnover . Mass spectrometry imaging combined with AGP26.10 immunolabeling could enable spatial proteomic analysis, correlating gp210 distribution with other proteins across tissues or subcellular regions without requiring multiple antibodies simultaneously . Proximity labeling techniques such as BioID or APEX could be combined with AGP26.10 immunoprecipitation to identify proteins in close proximity to gp210 under different physiological or experimental conditions, expanding our understanding of nuclear pore complex interaction networks . Cryo-electron tomography with immunogold labeling using AGP26.10 could provide unprecedented structural details of gp210 organization within the native nuclear pore complex in a near-physiological state . Finally, combining AGP26.10 with quantitative approaches like mass cytometry (CyTOF) could enable high-throughput analysis of gp210 expression across large numbers of cells while simultaneously measuring dozens of other cellular parameters, potentially revealing unexpected correlations between nuclear pore complex composition and cellular states .