ATL31 Antibody

What is ATL31 and why would researchers develop antibodies against it?

ATL31 is a ubiquitin ligase that functions as a regulator of carbon/nitrogen (C/N) nutrient stress responses in Arabidopsis. It contains an N-terminal transmembrane domain required for its function and localizes to multiple organelles including the plasma membrane, TGN/EE, and late endosomal compartments . Researchers would develop antibodies against ATL31 to study its endogenous expression, subcellular localization, protein interactions, and modifications without relying solely on tagged versions that might affect protein function.

What epitopes should be targeted when generating ATL31 antibodies?

When designing ATL31 antibodies, researchers should consider avoiding the transmembrane domain, which might be inaccessible in its native state. Instead, target unique regions in the cytosolic portion of the protein, particularly conserved regions of the RING domain (avoiding the catalytic cysteine-143 residue which is critical for function) or unique sequences in the N-terminal region . Peptide antigens derived from these regions would likely generate antibodies with high specificity and low cross-reactivity with other ATL family members.

What experimental validation should be performed when characterizing a new ATL31 antibody?

A newly developed ATL31 antibody should undergo rigorous validation including: (1) Western blot analysis comparing wild-type plants with ATL31 overexpression lines and atl31 knockouts; (2) immunoprecipitation followed by mass spectrometry to confirm target specificity; (3) immunofluorescence microscopy to verify subcellular localization patterns that match those observed with fluorescently-tagged ATL31; and (4) pre-absorption controls to demonstrate specificity . These validation steps ensure the antibody recognizes endogenous ATL31 with high specificity.

How can ATL31 antibodies complement GFP fusion studies for subcellular localization analysis?

While ATL31-GFP fusion studies have revealed localization to the plasma membrane and endosomal compartments, antibodies against native ATL31 can provide important verification that the GFP tag doesn't alter trafficking or localization patterns . For immunolocalization, researchers should use fixation methods that preserve membrane structures, co-stain with established organelle markers (such as mRFP-SYP43, mRFP-SYP61, VHAa1-mRFP for TGN/EE), and compare results with ATL31-GFP localization patterns to validate findings. This dual approach eliminates potential artifacts from overexpression or GFP fusion.

What fixation and sample preparation methods are optimal for ATL31 immunolocalization in plant tissues?

For optimal ATL31 immunolocalization in plant tissues, researchers should use a paraformaldehyde-based fixation (typically 4%) that preserves membrane structures while maintaining antigen accessibility. Given ATL31's membrane association, a mild detergent permeabilization step (0.1-0.5% Triton X-100) would be necessary after fixation . For root tissues, where ATL31 localization has been well-characterized, vibratome sectioning at 50-100 μm thickness would maintain cellular architecture while allowing antibody penetration. Antigen retrieval might be necessary if fixed samples show weak signal.

How can ATL31 antibodies be used to analyze redistribution under different C/N conditions?

To study ATL31 redistribution under various carbon/nitrogen conditions, researchers should treat Arabidopsis seedlings with media containing different glucose/nitrogen ratios (e.g., 0 mM Glc/60 mM N, 100 mM Glc/30 mM N, or 200 mM Glc/0.3 mM N) for 90-110 minutes . Following treatment, tissues can be fixed and processed for immunolocalization using ATL31 antibodies along with organelle markers. Quantitative analysis of colocalization coefficients between ATL31 and various organelle markers under different C/N conditions would reveal condition-dependent trafficking patterns. This approach would complement the fluorescent protein-based observations showing differential association with late endosomal markers under high C/low N conditions.

How can ATL31 antibodies be used to study native protein-protein interactions?

ATL31 antibodies can be employed for co-immunoprecipitation of native protein complexes from Arabidopsis tissues to identify physiologically relevant interaction partners. The protocol should include: (1) tissue extraction in a mild detergent buffer (e.g., 50 mM Tris-HCl pH 7.5, 0.5% Triton X-100, 150 mM NaCl, 10% glycerol, 1 mM EDTA) with protease inhibitors; (2) immunoprecipitation with ATL31 antibodies; (3) western blot analysis using antibodies against suspected interaction partners (e.g., SYP61) . This approach would validate interactions previously identified using tagged proteins and potentially identify new interaction partners under native expression conditions.

What controls are essential when studying ATL31-SYP61 interactions using antibody-based methods?

When investigating ATL31-SYP61 interactions using antibody-based approaches, several controls are critical: (1) use of atl31 knockout/knockdown plants to confirm specificity; (2) inclusion of a catalytically inactive ATL31 mutant (ATL31 C143S) to distinguish between binding and ubiquitination-dependent interactions; (3) use of pre-immune serum as a negative control for non-specific binding; and (4) competition with excess recombinant ATL31 protein to verify antibody specificity . For analyzing SYP61 interaction specifically, reciprocal co-immunoprecipitation (using SYP61 antibodies to pull down ATL31) provides additional validation.

How can researchers use ATL31 antibodies to study dynamic changes in protein interactions during C/N stress responses?

To investigate dynamic changes in ATL31 protein interactions during C/N stress responses, researchers should: (1) treat Arabidopsis seedlings with different C/N media compositions for varying durations; (2) perform co-immunoprecipitation with ATL31 antibodies; (3) analyze interaction partners by western blotting or mass spectrometry; and (4) quantify relative interaction strengths across conditions . This approach would reveal how ATL31 interaction networks change dynamically in response to nutritional stress. Time-course experiments would be particularly valuable to track the temporal sequence of interaction changes following nutrient stress application.

How can ATL31 antibodies be used to study endogenous ubiquitination activities?

To study ATL31-mediated ubiquitination of endogenous substrates, researchers should: (1) immunoprecipitate putative substrates (e.g., SYP61) from plant tissues; (2) analyze ubiquitination status by western blotting with anti-ubiquitin antibodies; (3) compare ubiquitination patterns between wild-type, ATL31 overexpression, and atl31 knockout plants; and (4) use linkage-specific anti-ubiquitin antibodies (e.g., anti-K63) to determine ubiquitin chain types . This approach would extend in vitro findings showing that ATL31 can catalyze K63-linked ubiquitination of SYP61. Researchers might also perform in vitro ubiquitination assays using immunopurified native ATL31 to verify its enzymatic activity.

What methodological approaches can confirm SYP61 as a physiological ubiquitination target of ATL31?

To confirm SYP61 as a physiological ubiquitination target of ATL31, researchers should implement multiple complementary approaches: (1) in vivo ubiquitination assays in Arabidopsis plants expressing tagged ubiquitin; (2) comparison of SYP61 ubiquitination levels between wild-type, ATL31 overexpression, and atl31 mutant plants; (3) mass spectrometry analysis to identify specific lysine residues on SYP61 that are ubiquitinated; and (4) mutagenesis of these lysine residues to confirm functional significance . Additionally, researchers should examine whether SYP61 stability or trafficking is altered in ATL31 mutants, providing functional evidence for the relevance of this ubiquitination.

How can researchers differentiate between different types of ubiquitin linkages mediated by ATL31?

To differentiate between ubiquitin linkage types catalyzed by ATL31, researchers should: (1) perform in vitro ubiquitination assays with recombinant ATL31 and analyze products using linkage-specific antibodies; (2) use targeted mass spectrometry with Ub-AQUA peptides for quantitative analysis of different linkage types; (3) employ ubiquitin mutants with specific lysine-to-arginine substitutions to determine which lysines are used for chain formation; and (4) compare results from in vitro assays with in vivo ubiquitination patterns . Parallel reaction monitoring mass spectrometry, as mentioned in the research, has shown that ATL31 can catalyze various ubiquitin linkages including K63, K11, and K48, suggesting complex signaling capabilities.

How can ATL31 antibodies be used in combination with membrane trafficking inhibitors?

Researchers can combine ATL31 immunolocalization with membrane trafficking inhibitors to dissect trafficking pathways. This approach would involve: (1) treating Arabidopsis seedlings with inhibitors such as Brefeldin A (BFA, 50 μM), wortmannin (WM, 33 μM), or Concanamycin A (ConcA, 1 μM); (2) performing immunolocalization with ATL31 antibodies; and (3) co-staining with organelle markers . BFA would induce aggregation of endosomal compartments into BFA bodies, WM would cause swelling of late endosomes, and ConcA would inhibit vacuolar degradation of ATL31. Analysis of ATL31 redistribution under these conditions would provide insights into its trafficking pathways.

What methodological considerations are important when studying ATL31 degradation using antibodies?

When investigating ATL31 degradation pathways, researchers should: (1) track endogenous ATL31 protein levels under various conditions using quantitative western blotting; (2) compare with GFP fluorescence patterns in transgenic ATL31-GFP plants; (3) block protein synthesis with cycloheximide to measure protein half-life; and (4) use inhibitors of different degradation pathways (proteasome inhibitors like MG132 or vacuolar degradation inhibitors like ConcA) . The search results indicate that ATL31 undergoes vacuolar degradation, which can be visualized by GFP fluorescence in vacuoles after dark treatment and confirmed by ConcA treatment, which enhances the ratio of full-length ATL31-GFP to free GFP.

How can ChIP-based approaches using ATL31 antibodies provide insights into potential nuclear functions?

While the current literature primarily describes ATL31 as a membrane-associated protein, many signaling proteins shuttle between membranes and the nucleus. To investigate potential nuclear functions, researchers could: (1) perform cellular fractionation followed by western blotting with ATL31 antibodies to detect nuclear pools; (2) if nuclear localization is detected, conduct chromatin immunoprecipitation (ChIP) using ATL31 antibodies to identify potential DNA binding sites; and (3) couple ChIP with sequencing (ChIP-seq) to generate genome-wide binding profiles . This experimental approach would reveal whether ATL31 might have direct roles in transcriptional regulation in addition to its characterized membrane trafficking and ubiquitination functions.

What extraction methods are optimal for maintaining ATL31 integrity in different experimental contexts?

For optimal ATL31 extraction, researchers should consider its membrane association and potential for degradation. The recommended protocol includes: (1) rapid tissue harvesting and flash-freezing in liquid nitrogen; (2) grinding tissue to a fine powder with stainless steel beads; (3) extraction in buffer containing 50 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 150 mM NaCl, 10% glycerol, and 1 mM EDTA with protease inhibitors; and (4) gentle mixing followed by centrifugation to remove debris . For western blotting, adding SDS sample buffer directly to ground tissue preserves protein integrity. When studying ubiquitination, including deubiquitinase inhibitors (N-ethylmaleimide) is essential to prevent artificial deubiquitination during extraction.

What are common issues when immunoprecipitating membrane-associated proteins like ATL31?

Common challenges when immunoprecipitating membrane-associated proteins like ATL31 include: (1) insufficient solubilization leading to low yields; (2) non-specific binding to beads; (3) co-purification of irrelevant membrane proteins; and (4) disruption of native protein complexes . To address these issues, researchers should optimize detergent type and concentration (testing alternatives to Triton X-100 such as digitonin or CHAPS), include appropriate blockers in washing buffers, perform pre-clearing steps with protein A/G beads, and consider crosslinking approaches to stabilize transient interactions. Additionally, using denaturing conditions for ubiquitination studies can prevent deubiquitination during sample processing.

How should researchers validate western blot signals when working with ATL31 antibodies?

To validate ATL31 western blot signals, researchers should implement multiple controls: (1) include atl31 knockout/knockdown samples as negative controls; (2) use ATL31 overexpression lines as positive controls; (3) verify signal specificity through peptide competition experiments; (4) confirm expected molecular weight shifts in samples from plants expressing tagged versions of ATL31; and (5) include loading controls like tubulin . For quantitative western blotting, researchers should establish a linear detection range for their antibody, use technical and biological replicates, and employ appropriate normalization methods to account for loading variations across samples.

How can ATL31 antibodies be applied in transient expression systems like Nicotiana benthamiana?

For ATL31 antibody applications in Nicotiana benthamiana, researchers should: (1) establish a transient expression system using Agrobacterium tumefaciens infiltration as described in the search results; (2) co-express ATL31 with potential interaction partners; (3) perform co-immunoprecipitation followed by western blotting; and (4) conduct subcellular localization studies through immunofluorescence . The advantage of using N. benthamiana is the ability to rapidly test multiple constructs and protein combinations. When using this system, researchers should include appropriate controls such as the p19 silencing suppressor to enhance expression and compare results with stable Arabidopsis transformants to confirm biological relevance.

What considerations are important when adapting ATL31 antibody applications from Arabidopsis to crop species?

When adapting ATL31 antibody applications to crop species, researchers should: (1) perform sequence alignments to determine conservation of ATL31 epitopes across species; (2) test antibody cross-reactivity using protein extracts from the crop species of interest; (3) optimize extraction buffers and immunoprecipitation conditions for tissue-specific differences; and (4) validate subcellular localization patterns in the new species . If cross-reactivity is insufficient, researchers might need to develop new antibodies targeting conserved regions of ATL31 homologs in crop species. Studies in crops could reveal whether ATL31-mediated C/N sensing mechanisms are conserved across diverse plant lineages.