At2g19790 Antibody

What is At2g19790 and what role does it play in plant cells?

At2g19790 is an Arabidopsis thaliana gene that encodes the sigma (σ) subunit of the adaptor protein 4 (AP-4) complex, designated as AP4S. This protein functions as a critical component of the heterotetrameric AP-4 complex, which also includes beta (β), mu (μ), and other subunits that collectively facilitate vesicle-mediated protein trafficking in the endomembrane system . The AP-4 complex localizes primarily to the trans-Golgi network and functions in the selective transport of cargo proteins to target cellular compartments. Functional analysis of AP-4 complex interactors reveals enrichment in pathways related to "translation," "protein complex disassembly," and "xylem development," suggesting specialized roles beyond basic endomembrane trafficking . Mutations in At2g19790, such as the gfs6-1 mutant which contains a nucleotide substitution at the splicing donor site, result in disrupted vacuolar protein sorting, demonstrating its essential role in protein trafficking pathways .

How do AP-4 complex components differ from other adaptor protein complexes in plants?

The AP-4 complex represents one of several adaptor protein complexes in plant cells, each with distinct subunit compositions, localizations, and functional roles. Unlike AP-1 and AP-2 complexes that share certain subunits (such as AP1/2B1), the AP-4 complex maintains unique subunit composition, including the At2g19790-encoded σ-subunit . GO analysis reveals that AP-4 interactors are specifically enriched in terms related to "translation," "protein complex disassembly," and "xylem development," which distinguishes it from other AP complexes . For instance, AP-3 interactors show enrichment in "cytokinesis" and "ribosome biogenesis," while AP-5 associates with "lysosomal transport," "Golgi to vacuole transport," "chromosome organization," and "histone modifications" . These functional differences highlight the specialized roles of each adaptor complex within the plant endomembrane system, with AP-4 potentially coordinating protein synthesis with trafficking and developmental processes in specialized tissues.

What should researchers know about generating antibodies against the AP4S subunit?

When generating antibodies against the AP4S subunit (At2g19790 product), researchers must consider several critical factors to ensure specificity and functionality. First, epitope selection is crucial—targeting unique regions of the protein that do not share homology with other adaptor protein complex subunits will minimize cross-reactivity . Researchers should perform thorough sequence alignment analysis against other sigma subunits (AP1S, AP2S, AP3S, AP5S) to identify unique peptide regions for immunization. Second, the antibody production method must be carefully considered; both polyclonal and monoclonal approaches have merit, with monoclonal antibodies offering higher specificity but potentially limited epitope recognition .

Before proceeding to experimental applications, comprehensive antibody characterization is essential, including structural integrity assessment via SDS-PAGE, IEF, HPLC, or mass spectrometry to confirm the antibody is not fragmented or aggregated . Specificity testing should include both positive controls (verified At2g19790 protein samples) and negative controls (other AP complex subunits) to confirm selective binding . Additionally, cross-reactivity testing against human tissues should be conducted if the antibody will be used in heterologous systems . Potency assays measuring binding affinity and functional activity should be performed against a reference standard to establish lot-to-lot consistency for reproducible experimental results .

How should researchers design experiments to validate At2g19790 antibody specificity?

Designing rigorous validation experiments for At2g19790 antibodies requires multi-layered approaches to confirm specificity. A comprehensive validation protocol should include the following methodological elements:

First, perform immunoblotting using wild-type Arabidopsis tissue lysates alongside positive and negative controls. Positive controls should include recombinant AP4S protein or overexpression lines, while negative controls should utilize ap4s mutant lines (such as gfs6-1) or other AP complex sigma subunit proteins to assess cross-reactivity . Expected results should show a single band at the predicted molecular weight (~17-20 kDa) in wild-type and overexpression samples, with absent or significantly reduced signal in mutant lines.

Second, conduct immunoprecipitation followed by mass spectrometry analysis to confirm that the antibody successfully captures the At2g19790 protein and its known interacting partners. The immunoprecipitated samples should contain the complete AP-4 complex components (β, μ, and ε subunits) in addition to AP4S . The protocol should include stringent washing steps and appropriate negative controls (isotype-matched irrelevant antibodies) to minimize false positives .

Third, perform immunolocalization studies using both confocal microscopy and subcellular fractionation to verify the expected trans-Golgi network localization pattern. Co-localization with established trans-Golgi markers should be evident, with minimal signal in other cellular compartments . Additionally, competitive binding assays using synthetic peptides corresponding to the immunizing epitope should abolish antibody binding, further confirming specificity . These comprehensive validation approaches provide multiple lines of evidence for antibody specificity and functionality in diverse experimental contexts.

What are the optimal extraction and sample preparation methods for detecting At2g19790 protein in plant tissues?

Optimal extraction and sample preparation for At2g19790 protein detection requires specialized protocols that preserve protein integrity while maximizing yield from plant tissues. The following methodological approach is recommended:

For protein extraction, use fresh young tissue (preferably developing leaves or seedlings) where endomembrane trafficking is highly active. Begin with a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, supplemented with protease inhibitor cocktail and phosphatase inhibitors (10 mM NaF, 1 mM Na₃VO₄) . This combination preserves membrane protein integrity while effectively solubilizing the AP-4 complex. For subcellular fractionation experiments, employ a sucrose gradient centrifugation approach to separate cellular compartments, focusing on the trans-Golgi network fraction where AP4S predominantly localizes .

Sample preparation for immunoblotting should avoid excessive heating; limit exposure to 70°C for 5 minutes in SDS sample buffer to prevent aggregation of membrane-associated proteins. For immunoprecipitation, pre-clear lysates with protein A/G beads for 1 hour at 4°C to reduce non-specific binding before antibody addition . When preparing samples for immunohistochemistry, opt for paraformaldehyde fixation (4%) for 30 minutes followed by gentle permeabilization with 0.1% Triton X-100 to maintain cellular architecture while allowing antibody access to intracellular targets.

For mass spectrometry analysis, perform in-solution digestion with a combination of Lys-C and trypsin after immunoprecipitation to achieve comprehensive peptide coverage. This approach increases the likelihood of detecting the signature peptides that uniquely identify the At2g19790 gene product among other adaptor complex proteins . Implement these methodological refinements to maximize sensitivity and specificity when working with At2g19790 antibodies across diverse experimental applications.

How can researchers effectively use At2g19790 antibodies for co-immunoprecipitation of AP-4 complex components?

Effective co-immunoprecipitation (co-IP) of AP-4 complex components using At2g19790 antibodies requires careful optimization of several key parameters. First, establish optimal antibody concentration through titration experiments, typically starting with 2-5 μg of antibody per 500 μg of total protein extract . Covalently cross-link the antibody to magnetic protein A/G beads using bis(sulfosuccinimidyl)suberate (BS3) or similar crosslinkers to prevent antibody co-elution and contamination of the final sample.

The lysis buffer composition significantly impacts co-IP efficiency. Use a buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% sodium deoxycholate, supplemented with protease inhibitors and 10% glycerol to stabilize protein complexes . This gentler detergent combination preserves protein-protein interactions within the AP-4 complex better than more stringent detergents like SDS. Perform lysis and all binding steps at 4°C with gentle rotation (8-10 rpm) to maintain complex integrity.

For elution, compare specific peptide elution (using the immunizing peptide) against low-pH glycine elution (50 mM glycine, pH 2.5) to determine which method yields the most intact complex with minimal antibody contamination. When analyzing co-IP results, implement parallel detection strategies for all four AP-4 subunits (β, μ, σ, and ε) to confirm successful capture of the intact complex . This can be accomplished through multiplexed western blotting or mass spectrometry analysis using parallel reaction monitoring (PRM) for targeted detection of signature peptides from each subunit.

The following table summarizes the expected interaction partners that should co-immunoprecipitate with At2g19790/AP4S based on current interactome data:

Successful co-IP experiments should recover all core AP-4 components with similar stoichiometry, confirming antibody functionality for complex-level studies .

What are common pitfalls in At2g19790 antibody-based experiments and how can they be resolved?

Researchers frequently encounter several challenges when working with At2g19790 antibodies that can compromise experimental outcomes. These pitfalls, along with targeted solutions, include:

High background signal in immunoblotting often results from insufficient blocking or non-specific antibody binding. To address this, implement a sequential blocking strategy using 5% non-fat milk followed by 2% BSA in TBS-T for 1 hour each . Additionally, pre-absorb the antibody with plant extract from ap4s knockout mutants to remove antibodies recognizing off-target epitopes. Titrating primary antibody concentrations (starting from 1:1000 and adjusting as needed) and extending wash times (4 × 15 minutes with TBS-T) can dramatically improve signal-to-noise ratios.

Inconsistent immunoprecipitation efficiency is another common challenge, often due to complex instability. Stabilize the AP-4 complex by incorporating protein crosslinkers like DSP (dithiobis(succinimidyl propionate)) at 0.5-2 mM for 30 minutes before lysis, followed by quenching with Tris buffer . This reversible crosslinking preserves transient interactions within the complex during extraction and immunoprecipitation procedures.

False-negative results in immunolocalization studies frequently occur when epitopes are masked within the native protein structure. Implement multiple antigen retrieval methods in parallel experiments, including heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) and enzymatic retrieval with proteinase K (1-5 μg/ml for 10-15 minutes) . Compare results across methods to identify optimal conditions that maintain cellular architecture while exposing the target epitope.

Cross-reactivity with other AP complex sigma subunits can compromise experiment specificity. Address this by performing parallel immunodepletions with recombinant AP1S, AP2S, AP3S, and AP5S proteins before using the antibody in critical experiments . This pre-clearing step removes antibodies with affinity for related proteins, leaving only those specific to AP4S. Additionally, validate all findings using genetic approaches, comparing antibody signal patterns in wild-type, heterozygous, and homozygous ap4s mutant backgrounds to confirm signal specificity .

How can researchers differentiate between specific and non-specific signals when using At2g19790 antibodies?

Differentiating between specific and non-specific signals when using At2g19790 antibodies requires implementation of rigorous controls and analytical approaches. First, always include genetic controls in experimental designs—wild-type, heterozygous, and homozygous ap4s mutant samples should show decreasing signal intensity proportional to gene dosage . The gfs6-1 mutant or T-DNA insertion lines targeting At2g19790 serve as excellent negative controls for validating signal specificity .

Perform antibody competition assays where the primary antibody is pre-incubated with excess immunizing peptide (50-100 fold molar excess) before application to samples . Specific signals should be significantly reduced or eliminated in competition-treated samples, while non-specific signals persist. This approach is particularly valuable for validating immunohistochemistry and immunofluorescence results.

For immunoblotting applications, implement two-color detection systems where the At2g19790 antibody is detected in one channel (e.g., green) and a known AP-4 complex partner is detected in another channel (e.g., red) . Specific signals should demonstrate co-localization, appearing yellow in merged images, while non-specific signals will appear only in the single channel. Additionally, compare signal patterns across multiple antibody lots and ideally across antibodies raised against different epitopes of the same protein—specific signals should remain consistent despite these variations.

Quantitative assessment using pixel intensity ratio analysis between regions of interest and background areas can establish objective thresholds for signal specificity. Generally, specific signals should exhibit signal-to-background ratios exceeding 3:1 . The following decision matrix summarizes criteria for determining signal specificity:

Implementing these analytical approaches systematically increases confidence in experimental results and minimizes misinterpretation of antibody-generated data .

How should researchers interpret contradictory results between At2g19790 antibody data and genetic data?

When faced with contradictory results between At2g19790 antibody data and genetic findings, researchers should implement a systematic analytical framework to reconcile these discrepancies. First, critically evaluate the antibody validation data—an incompletely validated antibody may detect related proteins or non-specific targets . Verify that the antibody has been tested against AP4S knockout tissues and assessed for cross-reactivity with other adaptor complex sigma subunits through immunoblotting or ELISA .

Consider post-translational modifications or alternative splicing events that might affect epitope availability or protein function without altering gene expression. The gfs6-1 mutant, for example, contains a nucleotide substitution at the splicing donor site, potentially generating abnormal transcript variants that could produce truncated or altered proteins still detectable by certain antibodies . Employ RNA-seq and proteomic analyses to characterize all transcript and protein variants present in your experimental system.

Evaluate the potential for genetic compensation—related adaptor complex components might be upregulated in response to At2g19790 mutation, partially rescuing phenotypes while complicating antibody-based detection . Perform comprehensive expression profiling of all adaptor complex components in wild-type versus mutant backgrounds to identify compensatory changes.

Temporal considerations may also explain discrepancies; protein persistence after genetic manipulation could result in a lag between genetic intervention and protein depletion. Implement time-course studies following genetic manipulation to track protein dynamics . Additionally, consider spatial regulation differences—tissue-specific or subcellular localization changes might reconcile seemingly contradictory whole-organism or whole-tissue results.

When discrepancies persist, implement orthogonal approaches that don't rely on antibody specificity, such as CRISPR-based tagging of endogenous At2g19790 with fluorescent proteins or proximity labeling techniques like TurboID . These approaches provide independent validation of protein localization and interaction patterns. Document all reconciliation attempts thoroughly, as apparent contradictions often lead to new biological insights about protein function, regulation, or the limitations of current experimental tools.

How can researchers use At2g19790 antibodies to study dynamic changes in AP-4 complex assembly?

Studying dynamic changes in AP-4 complex assembly using At2g19790 antibodies requires sophisticated experimental approaches that capture temporal and spatial aspects of complex formation. Implement pulse-chase immunoprecipitation experiments by metabolically labeling newly synthesized proteins with azidohomoalanine (AHA), followed by sequential immunoprecipitation with At2g19790 antibodies at defined time intervals . This approach reveals the kinetics of AP4S incorporation into the complete AP-4 complex and identifies any assembly intermediates.

For spatiotemporal analysis, combine At2g19790 antibodies with proximity labeling techniques such as TurboID or APEX2 fused to other AP-4 components . After brief activation of the proximity labeling enzyme, perform immunoprecipitation with the At2g19790 antibody followed by streptavidin pulldown to isolate proteins that were in proximity to the AP-4 complex at specific cellular locations and time points. This approach captures transient interactions that might be missed by standard co-immunoprecipitation methods.

Fluorescence fluctuation spectroscopy techniques, such as Number and Brightness (N&B) analysis or Fluorescence Correlation Spectroscopy (FCS), can be applied after immunofluorescent labeling with At2g19790 antibodies to measure oligomerization states of the AP-4 complex in different cellular compartments . These approaches provide quantitative data on complex stoichiometry and assembly dynamics with high spatial resolution.

To study stress-induced changes in AP-4 complex assembly, expose plant tissues to relevant stressors (salt, drought, pathogen elicitors) before performing quantitative immunoprecipitation with At2g19790 antibodies. Use mass spectrometry with tandem mass tags (TMT) or isobaric tags for relative and absolute quantitation (iTRAQ) to measure changes in the stoichiometry of AP-4 components and associated proteins under different environmental conditions . This approach reveals how external stimuli modulate AP-4 complex composition and potential cargo selection, providing insights into the adaptive trafficking responses mediated by this complex.

What approaches can be used to identify new cargo proteins recognized by the At2g19790-containing AP-4 complex?

Identifying novel cargo proteins recognized by the At2g19790-containing AP-4 complex requires multifaceted approaches that capture both stable and transient interactions. Begin with proximity-dependent biotin identification (BioID or TurboID) by fusing the biotin ligase to AP4S or other AP-4 complex components . After expression in plant cells and biotin supplementation, perform streptavidin pulldown followed by mass spectrometry to identify proteins that come into proximity with the AP-4 complex, potentially including cargo proteins that may only transiently interact with the complex during sorting.

Implement quantitative proteomic analysis of secretory pathway compartments in wild-type versus ap4s mutant plants to identify proteins whose subcellular localization is altered in the absence of functional AP-4 complex . Focus particularly on proteins showing aberrant accumulation in the trans-Golgi network or mislocalization to other compartments, as these represent potential cargo dependents on AP-4-mediated sorting.

For direct interaction studies, perform crosslinking immunoprecipitation (CLIP) using chemical crosslinkers like DSP or formaldehyde before immunoprecipitation with At2g19790 antibodies . This approach stabilizes transient interactions between the AP-4 complex and potential cargo proteins. Follow with stringent washes and mass spectrometry analysis to identify crosslinked proteins. Compare these results with immunoprecipitation performed without crosslinking to distinguish between stable complex components and transient cargo interactions.

Bioinformatic prediction can complement experimental approaches by analyzing protein sequences for potential AP-4 binding motifs. Based on established cargo recognition principles in mammalian systems, scan the Arabidopsis proteome for proteins containing similar motifs and prioritize these candidates for experimental validation using the At2g19790 antibody. The following workflow integrates these approaches for comprehensive cargo identification:

• Perform parallel BioID/TurboID labeling using AP4S-fusion proteins

• Conduct quantitative proteomics comparing subcellular fractions from wild-type and ap4s mutants

• Identify candidate cargo proteins present in both datasets

• Confirm direct interactions using crosslinking immunoprecipitation with At2g19790 antibodies

• Validate trafficking defects of candidate cargoes in ap4s mutant backgrounds

This integrated approach maximizes the probability of identifying biologically relevant AP-4 cargo proteins while minimizing false positives from each individual method .

How can At2g19790 antibodies be used to investigate AP-4 complex function across different plant developmental stages?

Investigating AP-4 complex function across developmental stages requires strategic application of At2g19790 antibodies within a developmental biology framework. Begin by establishing a comprehensive expression atlas through immunoblotting and immunohistochemistry using At2g19790 antibodies on tissues representing key developmental transitions: seed germination, seedling development, vegetative growth, reproductive transition, flowering, and senescence . Quantify expression levels and subcellular distribution patterns at each stage, correlating these with known developmental markers.

Implement tissue-specific immunoprecipitation followed by mass spectrometry to characterize stage-specific interactomes of the AP-4 complex . This approach reveals how AP4S/At2g19790 associations change throughout development, potentially indicating shifts in cargo selectivity or trafficking pathways. Use stable isotope labeling (such as SILAC or iTRAQ) for quantitative comparison across developmental stages, focusing on proteins that show significant association changes.

For functional analysis, develop co-immunofluorescence protocols using At2g19790 antibodies alongside markers for various endomembrane compartments (TGN, prevacuolar compartment, vacuole) across developmental stages . This approach reveals developmental shifts in AP-4 localization and potential changes in trafficking routes. Combine with super-resolution microscopy techniques like Structured Illumination Microscopy (SIM) or Stimulated Emission Depletion (STED) to visualize fine-scale changes in AP-4 distribution relative to endomembrane organization.

To directly link developmental phenotypes with AP-4 function, perform developmental stage-specific complementation experiments in ap4s mutant backgrounds . Express the At2g19790 gene under stage-specific promoters and use the antibody to confirm proper expression and localization of the complementing protein. This approach isolates the developmental windows where AP-4 function is critical and correlates molecular complex formation with developmental outcomes.

The following experimental matrix summarizes this developmental approach:

This comprehensive developmental approach reveals how the AP-4 complex dynamically adjusts its composition, location, and functional interactions to support changing cellular priorities throughout the plant life cycle .

How might new antibody technologies improve At2g19790/AP4S research?

Emerging antibody technologies offer significant opportunities to advance At2g19790/AP4S research beyond current limitations. Single-domain antibodies (nanobodies) derived from camelid immunoglobulins represent a promising frontier due to their small size (~15 kDa) and ability to recognize epitopes inaccessible to conventional antibodies . Developing At2g19790-specific nanobodies would enable live-cell imaging of AP-4 dynamics with minimal steric hindrance, potentially revealing trafficking events currently obscured by fixation artifacts or antibody size limitations.

Recombinant antibody engineering approaches, particularly the development of bispecific antibodies targeting both At2g19790 and other AP-4 components simultaneously, could enable selective immunoprecipitation of intact complexes while excluding partial assemblies . This technology would facilitate the purification of functional AP-4 complexes for structural studies and in vitro reconstitution experiments, addressing fundamental questions about complex assembly and cargo recognition mechanisms.

Antibody-based proximity labeling represents another promising direction, where At2g19790 antibodies are conjugated directly to enzymes like APEX2, HRP, or TurboID . When introduced into semi-permeabilized cells or applied to tissue sections, these conjugates would catalyze biotin labeling specifically in the vicinity of the AP-4 complex, enabling spatial proteomic mapping of the AP-4 microenvironment with unprecedented resolution. This approach would overcome limitations of overexpression-based proximity labeling systems that may disrupt complex stoichiometry or localization.

Developing stimuli-responsive antibody systems, where At2g19790 antibody binding or release can be triggered by light, temperature, or chemical inputs, would enable acute disruption of AP-4 function in specific subcellular locations . This optogenetic-like approach would provide temporal control over AP-4 complex integrity, allowing researchers to dissect the immediate consequences of complex disruption without the compensatory adaptations that often occur in genetic knockout systems.

These emerging antibody technologies, combined with advances in imaging, proteomics, and structural biology, position At2g19790/AP4S research for significant breakthroughs in understanding specialized trafficking pathways in plant cells.

What are promising research directions for understanding AP4S function beyond its role in the AP-4 complex?

While At2g19790/AP4S is primarily recognized as a component of the AP-4 adaptor complex, emerging evidence suggests potential moonlighting functions that warrant dedicated investigation. Several promising research directions could reveal AP4S functions beyond the canonical AP-4 complex:

Nuclear functions represent an intriguing possibility, as GO analysis of AP-5 interactors revealed unexpected associations with "chromosome organization" and "histone modifications," suggesting adaptor complex subunits might have nuclear roles . Researchers should employ chromatin immunoprecipitation (ChIP) with At2g19790 antibodies followed by sequencing to investigate potential DNA-binding activities or chromatin associations of AP4S. Fractionate nuclei from cytoplasmic components before immunoprecipitation to enrich for nuclear-specific interactions.

Unconventional protein-protein interactions outside the AP-4 complex merit exploration through large-scale yeast two-hybrid screens or proximity labeling in ap4b mutant backgrounds (where AP-4 complex cannot form but AP4S is still expressed) . This approach would reveal AP4S interactions that occur independently of the complete AP-4 complex. Particular attention should be paid to interactions with transcription factors, RNA-binding proteins, or signaling components that might indicate regulatory functions.

Potential roles in stress response pathways are suggested by the association of AP-2 interactors with "response to metal ion" terms . Similar stress-responsive functions might exist for AP4S. Researchers should quantify AP4S expression and localization under various abiotic stresses (drought, salt, heat, cold) and biotic challenges (bacterial, fungal, viral pathogens). Complementary proteomics approaches should identify stress-specific changes in the AP4S interactome that might reveal condition-specific functions.

Developmental regulation activities beyond trafficking are suggested by the association of AP-4 interactors with "xylem development" . Researchers should perform high-resolution immunohistochemistry with At2g19790 antibodies during vascular tissue differentiation, focusing on potential non-vesicular pools of AP4S. Combined with laser-capture microdissection and tissue-specific proteomics, this approach could reveal tissue-specific AP4S functions during developmental transitions.

These research directions challenge the conventional view of adaptor complex subunits as exclusively trafficking-related proteins and may reveal unexpected moonlighting functions that contribute to cellular homeostasis, development, or environmental adaptation.