ORP2A Antibody

What is ORP2A and why is it significant in plant research?

ORP2A is an Arabidopsis oxysterol-binding protein-related protein that mediates ER-autophagosomal membrane contact sites (EACS) and regulates autophagosome biogenesis. It localizes to both ER-plasma membrane contact sites (EPCSs) and autophagosomes, interacting with the ER-localized protein VAP27-1 via its FFATL motif and with autophagosome-located ATG8e. ORP2A is significant because it contributes to membrane dynamics during autophagy, binds multiple phospholipids including phosphatidylinositol 3-phosphate (PI3P), and plays roles in glucose signaling pathways by interacting with AtRGS1 . Unlike most autophagy-related mutants in plants, ORP2A knockdown causes severe developmental defects, suggesting broader functions beyond autophagy regulation.

What protein isoforms of ORP2A should researchers consider when selecting antibodies?

Researchers should be aware that ORP2A exists in two alternative protein forms resulting from different transcriptional start sites. Western blot analyses of flag-tagged ORP2A have confirmed this prediction from TAIR (The Arabidopsis Information Resource), showing two distinct bands: one around 100 kDa (full-length ORP2A) and another approximately 70 kDa (shortened version, designated ORP2AS) . When selecting or developing antibodies, researchers should consider whether their experimental goals require detection of both isoforms or specific targeting of one form. Epitope selection becomes particularly important, as antibodies targeting the N-terminal region might not detect both forms.

What immunodetection methods are most effective for ORP2A visualization?

For ORP2A detection, both direct immunodetection and tag-based methods have proven effective. In published research, flag-tagged ORP2A constructs (ORP2Ag-3×Flag) under native promoters have been successfully employed for Western blotting . For subcellular localization studies, fluorescent protein fusions (YFP-ORP2A) have been used effectively for live-cell imaging to identify ORP2A at membrane contact sites and autophagic structures . When using antibodies directly against ORP2A, standard Western blotting protocols with 8% SDS-PAGE gels have yielded clear results. For protein extraction, buffer compositions containing 50 mM Tris-HCL, 100 mM NaCl, 1% Triton-100, 10% Glycerol, 1mM PMSF, and protease inhibitor cocktail have been effective in preserving ORP2A integrity for immunodetection .

How can researchers distinguish between membrane-bound and soluble forms of ORP2A?

To distinguish between membrane-bound and soluble forms of ORP2A, researchers should employ differential centrifugation followed by immunoblotting. ORP2A localizes to both ER-plasma membrane contact sites and autophagosomes, making fractionation crucial for understanding its dynamic localization patterns . First, homogenize plant tissue in buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10% glycerol, and protease inhibitors. Then perform sequential centrifugation: low-speed (1,000g, 10 min) to remove nuclei and debris, medium-speed (20,000g, 20 min) to collect larger membrane fractions, and high-speed (100,000g, 1 hour) to separate microsomes from cytosolic fractions. After immunodetection of ORP2A in each fraction, compare with known markers: VAP27-1 for ER membranes, ATG8e for autophagosomal structures, and cytosolic markers. ORP2A's distribution pattern across these fractions will reveal its partitioning between membrane-associated and soluble states.

How can immunoprecipitation assays be optimized to study ORP2A protein complexes?

Immunoprecipitation (IP) of ORP2A requires careful optimization to preserve transient protein interactions at membrane contact sites. Based on published protocols for ORP2A interactions, researchers should use mild detergent conditions (0.1% Tween-20 rather than stronger detergents) to maintain membrane-associated complexes . For studying ORP2A complexes:

• Extract proteins from 200-300 mg of plant tissue using buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Tween-20, 10% glycerol, 1 mM PMSF, and protease inhibitor cocktail.

• Pre-clear lysates with protein A/G beads for 1 hour at 4°C to reduce nonspecific binding.

• Incubate cleared lysates with anti-ORP2A antibodies (or anti-tag antibodies for tagged constructs) coupled to beads overnight at 4°C with gentle rotation.

• Perform stringent washing (at least 3-5 washes) with buffer containing slightly increased salt concentration (150 mM NaCl) to remove non-specific interactions.

• Elute complexes under native conditions for subsequent activity assays or with SDS-sample buffer for immunoblotting.

Crosslinking with membrane-permeable reagents (e.g., DSP at 1-2 mM) prior to extraction can stabilize transient interactions between ORP2A and its partners like VAP27-1 and ATG8e .

What considerations are important when using ORP2A antibodies for detecting autophagy dynamics?

When using ORP2A antibodies to study autophagy dynamics, researchers must consider the transient nature of ORP2A's association with autophagic structures. Time-lapse imaging studies have shown that YFP-ORP2A punctae dynamically associate with mCh-ATG8e-labeled autophagosomes, appearing at autophagosome formation sites and then separating as the structure matures . Therefore, fixed-time-point immunostaining may capture only a subset of ORP2A-positive autophagosomal structures.

For accurate assessment of autophagy dynamics using ORP2A antibodies:

The table below summarizes expected ORP2A immunodetection patterns under different autophagy conditions:

How can researchers use ORP2A antibodies to investigate membrane contact site formation?

Investigating membrane contact sites with ORP2A antibodies requires specialized approaches because these structures are transient and occur at nanoscale proximity between two membranes. For effective immunodetection at these sites:

• Use super-resolution microscopy techniques (STORM, PALM, or SIM) combined with dual immunolabeling of ORP2A and its binding partners (VAP27-1 for ER, ATG8e for autophagosomes).

• Employ proximity ligation assays (PLA) to detect ORP2A interactions with VAP27-1 and ATG8e in situ, which generates fluorescent signals only when proteins are within 40 nm proximity.

• Perform sequential immunoelectron microscopy with gold particles of different sizes to visualize ORP2A at the ultrastructural level in relation to ER and autophagosomal membranes.

When analyzing ORP2A at membrane contact sites, it's critical to distinguish between ER-PM contact sites (EPCSs) and ER-autophagosomal contact sites (EACSs). Mutation studies have shown that the FFATL motif is essential for ORP2A's interaction with VAP27-1 and localization to EPCSs, while different regions mediate its interaction with ATG8e . Researchers can use the ORP2A mutant lacking the functional FFATL motif (ORP2A mut2F) as a control for antibody specificity in membrane contact site studies .

What are the methodological considerations when using ORP2A antibodies to investigate lipid binding and transfer?

ORP2A binds multiple phospholipids, particularly showing colocalization with phosphatidylinositol 3-phosphate (PI3P) in vivo . When using antibodies to study ORP2A's lipid binding functions:

• Combine immunoprecipitation with lipidomic analysis to identify lipids bound to ORP2A in different conditions. Extract lipids from immunoprecipitated ORP2A using a modified Bligh-Dyer method and analyze by mass spectrometry.

• Use fluorescently-labeled lipid probes (such as BODIPY-PI3P) in competition assays with purified ORP2A and detect binding shifts with ORP2A antibodies.

• Perform in vitro lipid transfer assays between donor and acceptor liposomes with purified ORP2A, assessing lipid movement using fluorescent lipid sensors.

• For in vivo studies, correlate ORP2A immunolocalization with fluorescent PI3P sensors to confirm colocalization patterns.

In ORP2A knockdown plants, PI3P distribution is altered, with enrichment in the ER membrane . Therefore, when analyzing lipid distribution in relation to ORP2A, researchers should compare PI3P patterns between wild-type and ORP2A-deficient plants. The PH domain of ORP2A mediates differential phosphatidyl phosphoinositide binding activity in vitro , making it an important region to consider when analyzing lipid interactions.

How can researchers address potential cross-reactivity issues with ORP2A antibodies?

ORP2A belongs to a family of oxysterol-binding protein-related proteins, with twelve members in Arabidopsis . This presents potential cross-reactivity challenges for antibodies. To address this:

• Perform validation experiments using ORP2A knockdown or knockout lines (such as the orp2a-1 mutant) as negative controls to confirm antibody specificity .

• Test antibody cross-reactivity against recombinant proteins from other ORP family members, particularly those with highest sequence homology.

• Use epitope mapping to identify unique regions in ORP2A for generating highly specific antibodies. The C-terminal region often provides better specificity than the more conserved OSBP domain.

• Consider using tagged ORP2A constructs under native promoters (pORP2A::ORP2Ag-3×Flag) as alternative approaches when antibody specificity cannot be conclusively verified .

When investigating specific functions of ORP2A in relation to membrane contact sites or autophagy, researchers should always include controls to differentiate ORP2A-specific effects from those potentially mediated by other ORP family members.

What is the optimal sample preparation protocol for preserving ORP2A subcellular localization?

Preserving ORP2A's native subcellular localization requires careful sample preparation, as its distribution between EPCSs, autophagosomes, and other compartments is dynamic and sensitive to extraction conditions:

• For immunofluorescence studies, use mild fixation with 2% paraformaldehyde for 20 minutes at room temperature, avoiding methanol fixation which can disrupt membrane structures.

• When permeabilizing cells, use 0.1% Triton X-100 or 0.05% saponin rather than stronger detergents that might disrupt membrane contact sites.

• For biochemical fractionation, maintain samples at 4°C throughout processing and use buffers containing 10% glycerol and protease inhibitors to stabilize membrane associations.

• Consider using cross-linking agents (0.5-1 mM DSP) prior to extraction to preserve transient protein-protein interactions at membrane contact sites.

The dynamic localization of ORP2A at autophagosome formation sites, as demonstrated by time-lapse imaging , means that synchronization of autophagy induction is crucial for consistent results in localization studies. Researchers should standardize their induction protocols (e.g., 2-4 hours of nitrogen starvation) to obtain reproducible ORP2A localization patterns.

How can conflicting data between ORP2A antibody detection and fluorescent protein fusions be reconciled?

Researchers may encounter discrepancies between native ORP2A localization (detected by antibodies) and fluorescent protein fusions. To reconcile such conflicts:

• Compare the localization patterns of N-terminal and C-terminal fluorescent protein fusions, as tag position can significantly affect protein trafficking and function.

• Validate fluorescent fusion functionality through complementation assays in ORP2A-deficient backgrounds (orp2a-1) to ensure the fusion proteins retain biological activity.

• Perform dual labeling experiments using both antibodies against native ORP2A and fluorescent tags to directly compare localization patterns.

• Consider potential overexpression artifacts when using constitutive promoters like 35S; use native promoters (pORP2A) for more physiologically relevant expression levels .

A particular challenge with ORP2A is its two alternative protein forms (100 kDa and 70 kDa) . Fluorescent protein fusions may not accurately represent both forms' distribution if the tag affects one isoform differently than the other. In such cases, isoform-specific antibodies or carefully designed tagging strategies recognizing both forms are necessary for comprehensive analysis.

How can ORP2A antibodies be used to investigate the relationship between autophagy and glucose signaling?

Recent research has identified ORP2A as a positive regulator of glucose signaling through its interaction with AtRGS1 (Regulator of G protein Signaling 1) . Investigating this dual role in both autophagy and sugar signaling requires specialized approaches using ORP2A antibodies:

• Perform co-immunoprecipitation studies under different glucose concentrations to assess how sugar availability affects ORP2A interactions with both autophagy machinery (ATG8e) and signaling components (AtRGS1).

• Use immunofluorescence to track changes in ORP2A localization during glucose-induced endocytosis of AtRGS1, which is a key regulatory event in G-protein signaling.

• Compare ORP2A/AtRGS1/VAP27-1 complex formation in wild-type plants versus autophagy-deficient mutants to determine whether autophagy status influences glucose signaling pathways.

Transcriptome analysis of orp2a-1 and agb1-2 (arabidopsis g-protein beta 1) mutants revealed significant overlap in differentially expressed genes, with shared pathways involved in signal transduction and cellular response to chemical stimuli . This suggests that ORP2A antibodies could be valuable tools for investigating the molecular intersection between nutrient sensing, G-protein signaling, and autophagy regulation in plants.

What approaches can be used to study post-translational modifications of ORP2A?

ORP2A likely undergoes various post-translational modifications that regulate its function and localization. To investigate these modifications:

• Use phospho-specific antibodies or general phospho-protein detection methods following ORP2A immunoprecipitation to assess phosphorylation status under different conditions (nutrient availability, stress).

• Perform immunoprecipitation followed by mass spectrometry to identify specific modified residues and the nature of modifications (phosphorylation, ubiquitination, acetylation).

• Compare modification patterns between the two ORP2A isoforms (100 kDa and 70 kDa) to determine if they are differentially regulated.

• Develop antibodies specific to known or predicted modification sites to track their occurrence in various physiological contexts.

The G-protein signaling pathway involves numerous phosphorylation events, and ORP2A's interaction with AtRGS1 suggests it may be subject to similar regulatory modifications . Additionally, autophagy regulation often involves phosphorylation cascades, making post-translational modification analysis crucial for understanding ORP2A's multifunctional roles.

How can CRISPR/Cas9-engineered ORP2A variants be validated using antibodies?

CRISPR/Cas9 technology offers powerful approaches for studying ORP2A function through precise genomic modifications. When validating CRISPR-engineered ORP2A variants:

• Use Western blotting with ORP2A antibodies to confirm successful editing, comparing protein size, abundance, and isoform patterns between wild-type and edited plants.

• Perform immunolocalization to verify that modified ORP2A proteins maintain correct subcellular targeting or to characterize altered localization patterns resulting from specific mutations.

• Combine immunoprecipitation with interaction studies to assess how engineered mutations affect ORP2A's ability to bind partners like VAP27-1, ATG8e, and AtRGS1 .

• Use domain-specific antibodies to verify the integrity of particular regions (FFATL motif, PH domain) in partially modified variants.

When designing CRISPR strategies for ORP2A, researchers should consider the gene's alternative transcriptional start sites to ensure both protein isoforms are appropriately modified or preserved depending on experimental goals . Validation using antibodies that recognize epitopes outside the modified regions is essential for confirming that observed phenotypes result from specific engineered changes rather than unintended disruptions to ORP2A expression.