PATL4 Antibody
Description
Cellular Localization
Studies using PATL4-specific antibodies have revealed its dynamic localization:
-
Cell Plate Formation: Recruited during late cytokinesis stages, similar to PATL1 .
-
Subcellular Distribution: Associates with Golgi apparatus and endoplasmic reticulum membranes .
Functional Insights
Antibody-based assays have elucidated PATL4’s roles in:
-
Vesicle Trafficking: Mediates lipid transfer between membranes via Sec14 domain activity .
-
Cytokinesis Regulation: Localizes to cell plates during root development, suggesting a role in cell division .
-
Stress Responses: Elevated expression under phosphate starvation, implicating PATL4 in nutrient sensing .
Comparative Analysis of PATL Family Members
PATL4 shares functional overlap with other patellins but exhibits unique expression patterns:
| Protein | Expression Pattern | Key Function |
|---|---|---|
| PATL1 | Root cell plates | Cytokinesis, phosphoinositide binding |
| PATL3 | Outer root cell layers | Membrane trafficking |
| PATL4 | Vascular tissues, mitotic zones | Lipid signaling, stress adaptation |
Technical Considerations for PATL4 Antibody Use
-
Specificity: Antibodies must distinguish PATL4 from homologous proteins (e.g., PATL1/PATL2 share 88% Sec14 domain identity) .
-
Epitope Conservation: Cross-reactivity with orthologs in other plant species has not been validated.
-
Assay Compatibility: Works in immunoblotting (∼70 kDa band) and immunofluorescence but requires calcium-free buffers to avoid artifactual aggregation .
Unanswered Questions and Future Directions
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
PATL4 Antibody FAQs for Academic Researchers
How do PATL4 antibodies differ in specificity compared to other patellins (e.g., PATL1/PATL2)?
PATL4 shares high sequence similarity with homologs (e.g., PATL1/PATL2 share 88% identity in their Sec14 domains). To ensure specificity:
| Parameter | PATL4 | PATL1/PATL2 |
|---|---|---|
| Expression | Vascular tissues, mitotic zones | Root cell plates, outer layers |
| Function | Lipid signaling, stress adaptation | Phosphoinositide binding, cytokinesis |
| Antibody Validation | Requires pre-adsorption with recombinant PATL1/PATL2 to block cross-reactivity |
Advanced Strategy: Use peptide-blocking assays to confirm epitope uniqueness. For example, block PATL4 antibodies with synthetic peptides corresponding to its N-terminal region to rule out cross-reactivity.
What experimental controls are essential when using PATL4 antibodies?
To validate antibody performance:
-
Positive Controls: Use PATL4-overexpressing cell lines (e.g., Arabidopsis vascular tissue) or recombinant PATL4 protein.
-
Negative Controls: Include samples treated with PATL4 siRNA/shRNA knockdown to confirm loss of signal.
-
Buffer Optimization: Test calcium-free buffers (e.g., PBS without Ca²⁺/Mg²⁺) to prevent artifactual aggregation during IF.
Data Contradiction Handling: If conflicting results arise between IB and IF, verify glycosylation status. PATL4’s glycosylation may mask epitopes in certain assays, necessitating PNGase F treatment for deglycosylation.
How do PATL4 antibodies perform in nutrient stress models (e.g., phosphate starvation)?
PATL4 expression is upregulated under phosphate starvation, suggesting a role in nutrient sensing. Antibody-based studies reveal:
-
Localization Shifts: PATL4 redistributes to stress-induced membrane domains during phosphate deprivation.
-
Functional Assays: Co-staining with lipid markers (e.g., PIP2) can confirm PATL4’s role in membrane remodeling .
Advanced Protocol: Combine PATL4 antibodies with phospho-specific probes (e.g., anti-P-Tyr) to study signaling crosstalk during stress .
What are the limitations of current PATL4 antibodies, and how can they be addressed?
Key Limitations:
-
Cross-reactivity: High homology with PATL1/PATL2 requires stringent validation.
-
Ortholog Conservation: No validated cross-reactivity with non-Arabidopsis species.
-
Epitope Accessibility: Glycosylation may hinder detection in native tissues.
Solutions:
-
Epitope Mapping: Use peptide arrays to identify PATL4-specific epitopes in the N-terminal region.
-
Ortholog Testing: Screen antibodies against recombinant PATL4 from other species (e.g., Brassica) via ELISA.
-
Glycosidase Treatment: Pretreat lysates with PNGase F to resolve deglycosylated PATL4 in IB.
How can PATL4 antibodies be integrated into multi-omics workflows?
To study PATL4 in broader biological contexts:
-
Proteomics: Use PATL4 antibodies for immunoprecipitation (IP) coupled with mass spectrometry to identify interacting proteins (e.g., lipid transfer partners) .
-
Metabolomics: Combine PATL4 IF with lipid profiling (e.g., α-tocopherol quantification) to link localization to metabolic activity .
-
CRISPR Editing: Validate PATL4 knockout phenotypes using antibodies to confirm gene editing efficiency.
Example Workflow:
What advanced antibody engineering strategies can enhance PATL4 research?
To overcome limitations:
-
Single-Domain Antibodies (sdAbs): Engineered for smaller size and higher specificity in crowded membrane environments .
-
Bispecific Antibodies: Target PATL4 and phospholipids (e.g., PIP2) to study membrane remodeling dynamics .
-
Fluorescent Tagging: Fuse PATL4 with GFP for live-cell imaging to complement antibody-based studies.
Example Application: Use sdAbs to track PATL4 dynamics in real-time during cytokinesis, avoiding fixation artifacts.
How do PATL4 antibodies compare to other SEC14-GOLD protein antibodies (e.g., PATL1/PATL2)?
| Parameter | PATL4 Antibodies | PATL1/PATL2 Antibodies |
|---|---|---|
| Localization | Golgi/ER membranes, cell plates | Root cell plates, plasma membrane |
| Stress Response | Phosphate starvation | Salt stress, freezing |
| Lipid Binding | α-Tocopherol (indirect evidence) | Phosphoinositides (PATL1) |
Contradiction Note: PATL4’s role in lipid signaling is inferred from homology but requires direct validation via lipid-binding assays .
What computational tools can aid PATL4 antibody validation and epitope prediction?
-
Epitope Mapping: Use tools like EpiPred to predict linear epitopes in PATL4’s N-terminal region .
-
Ortholog Analysis: Align PATL4 sequences across species to identify conserved epitopes using BLAST or MUSCLE.
-
Structural Modeling: Predict PATL4-antibody interactions via Rosetta or AlphaFold if crystal structures are unavailable .
Example: Model PATL4’s Sec14 domain to identify surface-exposed residues for antibody targeting .
How can PATL4 antibodies inform therapeutic strategies in plant immunity or stress resilience?
While PATL4 is not a direct therapeutic target, antibodies can elucidate mechanisms for:
-
Enhancing Stress Tolerance: By studying PATL4-mediated lipid signaling under nutrient deprivation.
-
Biomarker Development: Identifying PATL4 expression patterns as indicators of vascular stress in crops.
-
Antioxidant Regulation: Linking PATL4 to vitamin E metabolism, as seen in PATL2 .
Future Direction: Engineer PATL4-overexpressing crops to test stress resilience, validated via antibody-based assays.
