At4g27120 Antibody

What is the At4g27120 gene and why are antibodies against it important for research?

At4g27120 encodes the Arabidopsis thaliana C53 protein (AtC53), a highly conserved cytosolic protein that functions as an ER-phagy receptor during endoplasmic reticulum stress. Antibodies against this protein are crucial for studying selective autophagy mechanisms, particularly ER-phagy pathways that maintain ER homeostasis during proteotoxic stress. These antibodies enable visualization and quantification of AtC53 in various experimental contexts, including its recruitment to autophagosomes during stress conditions . The conservation of C53 across plant and mammalian systems makes these antibodies valuable tools for comparative studies of fundamental autophagy mechanisms across kingdoms .

How do At4g27120 antibodies help distinguish between diffuse and punctate localization patterns?

At4g27120 antibodies are essential for monitoring the transition of AtC53 from its diffuse cytosolic pattern to punctate structures during ER stress. Under normal conditions, AtC53 displays a diffuse pattern partially overlapping with ER markers like GFP-HDEL, but upon ER stress induced by agents such as tunicamycin, AtC53 forms distinct puncta that represent its recruitment to autophagosomes . These antibodies can be used in both fixed-cell immunofluorescence and live-cell imaging with fluorescent protein fusions to quantify the number and intensity of puncta formation under various stress conditions . This localization shift serves as a crucial visual indicator of activated ER-phagy and requires proper antibody specificity to accurately distinguish between these patterns .

What controls should be included when using At4g27120 antibodies in autophagy studies?

When using At4g27120 antibodies for autophagy research, several essential controls must be implemented. First, include negative controls using autophagy-deficient mutants (such as atg5 and atg2) to confirm that the observed puncta formation is autophagy-dependent . Second, employ vacuolar degradation inhibitors (such as concanamycin A for plants or bafilomycin for mammalian cells) to confirm that the structures are destined for autophagic degradation . Third, use multiple inducers of ER stress (tunicamycin, DTT, CPA, phosphate starvation) to validate stress-specific responses . Fourth, perform parallel experiments with both antibody detection of endogenous AtC53 and fluorescently-tagged versions to rule out tagging artifacts . Finally, include wild-type specimens alongside experimental treatments to establish baseline expression and localization patterns .

How can At4g27120 antibodies be optimized for Western blot-based autophagic flux assays?

Optimizing At4g27120 antibodies for Western blot-based autophagic flux assays requires several methodological considerations. First, establish the appropriate antibody dilution through titration experiments to achieve optimal signal-to-noise ratio, typically starting with manufacturer recommendations and adjusting as needed. For detecting endogenous AtC53, samples should be collected at multiple time points after treatment with ER stressors (such as tunicamycin at 10 μg/ml) with and without autophagy inhibitors (concanamycin A at 1 μM for plants) . Apply equal protein loading (20-50 μg total protein) and verify with housekeeping controls. For optimal AtC53 detection, use 10-12% SDS-PAGE gels and PVDF membranes, followed by blocking with 5% non-fat dry milk . During quantification, normalize band intensities to loading controls and calculate the autophagic flux by comparing the ratio of accumulated protein in inhibitor-treated versus untreated samples under each stress condition . This methodology has successfully demonstrated that AtC53 autophagic flux is specifically induced during ER stress but not during carbon or nitrogen starvation conditions .

What immunoprecipitation protocols work best with At4g27120 antibodies for identifying interaction partners?

For optimal immunoprecipitation with At4g27120 antibodies to identify interaction partners, a multi-step protocol is recommended. Begin by crosslinking the antibody to magnetic beads (Protein A/G) using BS3 or similar crosslinkers to prevent antibody contamination in the final elution. For plant tissue, grind 5-10g of material in liquid nitrogen and extract proteins in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 2 mM EDTA, 5 mM DTT, 1% Triton X-100, protease inhibitor cocktail, and phosphatase inhibitors . Clear lysates by centrifugation at 20,000g for 20 minutes at 4°C. Pre-clear the lysate with naked beads before incubation with antibody-conjugated beads overnight at 4°C with gentle rotation . After binding, perform at least 5 washes with decreasing salt concentration buffers to maintain specific interactions. Elute bound proteins with acidic glycine buffer or by boiling in SDS sample buffer if the antibodies were not crosslinked . This approach has successfully identified key AtC53 interaction partners including UFL1 and DDRGK1, revealing the formation of a ternary receptor complex important for ER-phagy .

How can At4g27120 antibodies be used effectively in immunogold labeling for electron microscopy?

Effective immunogold labeling with At4g27120 antibodies for electron microscopy requires precise sample preparation and labeling protocols. Begin by fixing plant tissue in 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer (pH 7.2) for 2 hours at room temperature . After dehydration through an ethanol series, embed samples in LR White resin and prepare ultrathin sections (70-90 nm) on nickel grids. For immunolabeling, block non-specific binding sites with 2% BSA and 0.1% fish gelatin in PBS for 30 minutes . Incubate sections with primary At4g27120 antibody at 1:50 to 1:200 dilution overnight at 4°C, followed by thorough washing with PBS containing 0.1% Tween-20 . Apply gold-conjugated secondary antibodies (10-15 nm gold particles) at 1:30 dilution for 2 hours at room temperature. After washing, post-stain sections with uranyl acetate and lead citrate for contrast enhancement . This technique has successfully demonstrated that AtC53 is associated with ER membranes under normal conditions and is recruited to autophagosomes during ER stress, providing ultrastructural evidence for its role in selective autophagy .

How can At4g27120 antibodies be used to distinguish between different types of selective autophagy?

At4g27120 antibodies provide powerful tools for distinguishing between different selective autophagy pathways through careful experimental design. To differentiate ER-phagy from other forms of selective autophagy, implement a comparative approach exposing cells or tissues to multiple stress conditions: tunicamycin (ER stress inducer, 10 μg/ml), carbon starvation (bulk autophagy), nitrogen starvation (bulk autophagy), and phosphate starvation (mixed response) . Perform quantitative immunofluorescence or Western blot assays under each condition to track AtC53 recruitment to autophagosomes and degradation kinetics. AtC53 antibodies can be used alongside markers for other selective autophagy receptors in co-localization studies . The data will reveal that AtC53 puncta formation and degradation are specifically induced during ER stress but not during carbon or nitrogen starvation conditions that trigger bulk autophagy . This selectivity profile distinguishes ER-phagy from other forms of selective autophagy and provides insight into the specificity mechanisms of the autophagy machinery .

How can At4g27120 antibodies help elucidate the non-canonical ATG8 interaction mechanisms?

At4g27120 antibodies are instrumental in investigating the non-canonical ATG8 interaction mechanism known as the shuffled ATG8 interacting motif (sAIM). To elucidate these mechanisms, implement a multi-faceted approach combining in vitro and in vivo techniques. First, use purified recombinant proteins in pull-down assays with wild-type and mutant versions of AtC53 to map the interaction domains . Follow with co-immunoprecipitation experiments using At4g27120 antibodies in cells expressing various ATG8 isoforms to validate interactions in native contexts . For validating the non-canonical nature of the interaction, perform competition assays with synthetic peptides representing canonical AIM and shuffled AIM sequences, using isothermal titration calorimetry to measure binding affinities . To visualize the interaction in vivo, use proximity ligation assays or FRET with fluorescently tagged proteins. These approaches have successfully demonstrated that C53 interacts with ATG8 via a non-canonical shuffled AIM motif, revealing new mechanisms of selective autophagy substrate recognition that expand our understanding of the autophagy receptor code .

What are the critical factors for reproducing At4g27120 antibody-based autophagic flux assays across different plant species?

Reproducing At4g27120 antibody-based autophagic flux assays across different plant species requires careful consideration of several critical factors. First, assess antibody cross-reactivity with the C53 orthologs in target species through Western blot validation, as C53 is conserved but may have sequence variations that affect epitope recognition . Second, optimize protein extraction buffers for each species, considering differences in cell wall composition, secondary metabolites, and proteases that may interfere with extraction or protein stability . Third, adjust stress treatment conditions based on species-specific sensitivities; for example, tunicamycin concentrations may need to be optimized (range of 5-20 μg/ml) for different species based on pilot dose-response experiments . Fourth, establish appropriate autophagy inhibitor concentrations for each species (concanamycin A for plants, bafilomycin for mammalian cells) to accurately measure autophagic flux . Finally, identify species-specific reference genes for normalization in quantitative analyses. These methodological adjustments have successfully enabled comparative studies between Arabidopsis thaliana and Marchantia polymorpha, demonstrating the evolutionary conservation of C53-mediated autophagy mechanisms across plant lineages .

How can researchers troubleshoot non-specific binding when using At4g27120 antibodies in complex plant tissues?

When encountering non-specific binding with At4g27120 antibodies in complex plant tissues, implement a systematic troubleshooting approach. First, optimize blocking conditions by testing different blocking agents (5% BSA, 5% non-fat dry milk, commercial blocking reagents) and extending blocking times to 2-3 hours at room temperature . Second, increase the stringency of wash buffers by adjusting salt concentration (150-500 mM NaCl) and adding detergents like Tween-20 (0.1-0.3%) to reduce non-specific hydrophobic interactions . Third, pre-absorb the antibody with proteins extracted from c53 knockout mutant tissue to remove antibodies that recognize non-specific epitopes . Fourth, implement a dual-validation approach by comparing results from multiple antibody-based techniques (Western blot, immunofluorescence) with fluorescent protein fusions . Finally, include appropriate genetic controls (knockouts, knockdowns) in all experiments to definitively identify specific signals. This comprehensive approach has successfully addressed non-specific binding issues in various experimental contexts involving AtC53, enabling clear distinction between specific and non-specific signals .

How should researchers interpret contradictory results between antibody-detected endogenous At4g27120 and fluorescently-tagged fusion proteins?

When faced with contradictory results between antibody-detected endogenous At4g27120 and fluorescently-tagged fusion proteins, a structured analytical approach is essential. First, verify antibody specificity by confirming absence of signal in c53 knockout mutants. Second, evaluate whether the fluorescent tag affects protein function by performing complementation assays in c53 mutants with the tagged protein . Third, consider tag position effects by comparing N-terminal versus C-terminal fusions, as tags may mask interaction domains or targeting signals . Fourth, assess expression levels, as overexpression may lead to artifacts through altered stoichiometry with interaction partners like UFL1 and DDRGK1 . Fifth, examine the subcellular context by comparing results across multiple cellular compartments and stress conditions . Sixth, validate key findings with alternative approaches such as proximity labeling or mass spectrometry. Research has shown that native promoter-driven constructs better recapitulate endogenous behavior compared to constitutive promoters, which may explain some discrepancies between endogenous and overexpressed proteins . When properly validated, both approaches provide complementary insights into AtC53 function .

What statistical approaches are most appropriate for quantifying At4g27120 puncta formation in microscopy experiments?

Quantifying At4g27120 puncta formation in microscopy experiments requires robust statistical approaches to ensure reliable and reproducible results. Begin by establishing standardized image acquisition parameters, including consistent exposure times, Z-stack intervals, and resolution settings across all experimental conditions . For puncta quantification, use automated detection software with defined intensity thresholds and size parameters, validating these settings against manual counts in a subset of images . Normalize puncta counts to cell number or tissue area to account for variation in cell density or tissue thickness. For statistical analysis, collect data from at least 10 biological replicates per condition and perform appropriate statistical tests based on data distribution . For normally distributed data, use parametric tests (t-test for two conditions, ANOVA with post-hoc tests for multiple conditions); for non-parametric data, use Mann-Whitney or Kruskal-Wallis tests . Present results as mean ± standard deviation and include scatter plots to show distribution of individual data points . This rigorous quantitative approach has successfully demonstrated significant increases in AtC53 puncta formation specifically during ER stress conditions compared to carbon starvation or control conditions .

How can At4g27120 antibodies be applied in studying the ternary receptor complex during ER stress?

At4g27120 antibodies offer powerful tools for investigating the ternary receptor complex formed by C53, UFL1, and DDRGK1 during ER stress. To study this complex, implement a multi-level experimental approach. First, perform sequential co-immunoprecipitation using At4g27120 antibodies followed by UFL1 or DDRGK1 antibodies to isolate the intact complex from stressed and unstressed tissues . Second, apply proximity labeling techniques by fusing BioID or TurboID to AtC53 and using streptavidin pulldown followed by Western blot with At4g27120 antibodies to identify transient interaction partners . Third, use structured illumination or super-resolution microscopy with immunofluorescence to visualize the spatial organization of the complex components during stress induction . Fourth, implement sequential ChIP (chromatin immunoprecipitation) if any components have nuclear functions. Finally, compare complex formation across different stress types and intensities using quantitative proteomics. Research has demonstrated that the C53-UFL1-DDRGK1 complex is specifically formed during ER stress and is essential for recruiting specific ER components to autophagosomes, providing a mechanistic link between the ufmylation machinery and selective autophagy .

How can researchers use At4g27120 antibodies to investigate cross-talk between different selective autophagy pathways?

To investigate cross-talk between different selective autophagy pathways using At4g27120 antibodies, implement an integrated experimental strategy. First, perform co-immunoprecipitation with At4g27120 antibodies under various stress conditions (ER stress, oxidative stress, mitochondrial stress) followed by mass spectrometry to identify stress-specific interaction partners that may belong to different selective autophagy pathways . Second, use multiplexed immunofluorescence or proximity ligation assays to visualize co-localization of AtC53 with other selective autophagy receptors under different stress conditions . Third, generate plants with mutations in multiple autophagy receptor genes and perform epistasis analysis using At4g27120 antibodies to detect changes in localization or degradation patterns . Fourth, use quantitative Western blotting to measure changes in AtC53 levels when other selective autophagy pathways are activated or inhibited . This approach has revealed that while C53-mediated ER-phagy is distinct from bulk autophagy triggered by carbon or nitrogen starvation, it shares regulatory components with other selective autophagy pathways, including interaction with core autophagy machinery like ATG8 and ATG11/FIP200 . These findings provide insight into how cells coordinate different autophagy responses during complex stress scenarios .

What are the considerations for using At4g27120 antibodies in studying evolutionary conservation of ER-phagy mechanisms?

Using At4g27120 antibodies to study evolutionary conservation of ER-phagy mechanisms requires careful consideration of several factors. First, perform sequence alignment and epitope mapping of C53 orthologs across species to predict cross-reactivity of existing antibodies or design new ones targeting conserved regions . Second, validate antibody specificity in each species using knockout/knockdown controls and Western blotting to confirm recognition of appropriately sized proteins . Third, implement heterologous expression systems to test functional conservation, expressing fluorescently tagged orthologs in model systems alongside immunodetection with At4g27120 antibodies . Fourth, develop species-specific experimental conditions for stress induction and autophagy inhibition based on pilot experiments in each system . Fifth, use comparative proteomics with immunoprecipitation to identify conserved and divergent interaction partners across species. Research has demonstrated that C53's role in ER-phagy is conserved from plants to mammals, with human C53 interacting with mammalian ATG8 homologs GABARAP and GABARAPL1 similar to plant C53-ATG8 interactions . This evolutionary conservation suggests that C53-mediated ER-phagy represents an ancient selective autophagy mechanism that evolved before the divergence of plants and animals .