DRP1A Antibody

What is DRP1A and what cellular processes does it regulate in plants?

DRP1A (also known as ADL1, ADL1A, AG68, DL1, DYNAMIN-LIKE PROTEIN, DYNAMIN-RELATED PROTEIN 1A, RADIAL SWELLING 9, or RSW9) is a dynamin-like protein related to phragmoplastin in plants. This protein functions in multiple essential cellular processes including embryogenesis, cell plate formation during cytokinesis, trichome branching, and vascular patterning in coordination with VAN3 and GNOM . Significantly, DRP1A participates with DRP2B in clathrin-coated vesicle formation during endocytosis, making it a key component of the plant endocytic machinery . Research has demonstrated that DRP1A plays critical roles in plant immunity responses, particularly by regulating the PM-associated levels of the flagellin receptor FLS2, which is necessary for plants to initiate distinct immune signaling pathways .

How can researchers detect DRP1A protein in plant tissues?

The preferred methodology for DRP1A detection involves immunoblotting using specific anti-DRP1A antibodies. For optimal results, researchers should:

• Extract total proteins from plant tissues using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, and protease inhibitors

• Separate proteins via SDS-PAGE (10-12% gels work well for the ~68 kDa DRP1A)

• Transfer to PVDF or nitrocellulose membranes

• Block with 5% non-fat milk in TBST

• Incubate with anti-DRP1A primary antibody (typically at 1:1000-1:2000 dilution)

• Visualize using appropriate secondary antibody and detection system

For immunocytochemical analyses, fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 provides good results for subcellular localization studies . When probing for DRP1A in various plant species, researchers should consider cross-reactivity profiles, as the commercially available antibodies show reactivity with DRP1A from multiple plant species including Arabidopsis thaliana, Brassica species, Solanum species, and various cereal crops .

What are the most useful experimental controls when working with DRP1A antibody?

When conducting DRP1A antibody-based experiments, implementing appropriate controls is crucial for result validation:

Research has validated the specificity of anti-DRP1A antibodies by demonstrating absence of signal in drp1a mutants, while maintaining detection in wild-type controls . This type of genetic validation provides the strongest evidence for antibody specificity in plant research.

How can researchers use DRP1A antibody to investigate plant immune responses?

DRP1A plays significant roles in plant immunity, and antibody-based approaches offer powerful insights into these functions. To investigate DRP1A's role in plant immunity:

• Compare DRP1A protein levels in plants before and after pathogen exposure using quantitative immunoblotting

• Conduct co-immunoprecipitation with anti-DRP1A antibody to identify immune-related interaction partners

• Perform immunofluorescence microscopy to track DRP1A relocalization during immune responses

• Use proximity ligation assays with anti-DRP1A and antibodies against known immune components

Research has revealed that DRP1A contributes positively to pattern-triggered immunity (PTI) against bacterial pathogens like Pseudomonas syringae pv. tomato DC3000 . Immunoblotting analysis has shown that while drp1a mutants maintain normal steady-state levels of the immune receptor FLS2, they exhibit defects in ligand-induced receptor endocytosis . These findings suggest DRP1A regulates receptor dynamics rather than baseline expression, highlighting the value of temporal studies using anti-DRP1A antibodies.

How do DRP1A and DRP2B functionally interact and how can this interaction be studied?

DRP1A and DRP2B demonstrate functional overlap in endocytosis and immune signaling. To investigate their interactions:

• Perform sequential immunoprecipitation with both anti-DRP1A and anti-DRP2B antibodies

• Conduct immunofluorescence co-localization studies using antibodies against both proteins

• Apply proximity-dependent biotinylation (BioID) coupled with immunodetection

• Analyze protein dynamics in drp1a, drp2b, and drp1a drp2b double mutants

Research has demonstrated that DRP1A and DRP2B exhibit subcellular co-localization and direct intermolecular interactions . Both proteins contribute to clathrin-mediated endocytosis, with drp1a mutants showing more severe defects in ligand-induced endocytosis of the FLS2 receptor compared to drp2b mutants . Comparative immunoblotting of single and double mutants has revealed that DRP1A protein is undetectable in drp1a single and drp1a drp2b double mutants, providing important genetic tools for studying these interactions .

What methodologies can distinguish between different post-translational modifications of DRP1A protein?

DRP1A function is regulated by various post-translational modifications. To differentiate between these modifications:

• Use phospho-specific antibodies that recognize specific phosphorylation sites

• Employ Phos-tag™ SDS-PAGE followed by anti-DRP1A immunoblotting

• Conduct 2D gel electrophoresis (separating by isoelectric point and molecular weight) with anti-DRP1A detection

• Perform mass spectrometry analysis following DRP1A immunoprecipitation

While specific information about DRP1A phosphorylation in plants is limited in the provided search results, studies on mammalian Drp1 have demonstrated the importance of phosphorylation at specific sites (e.g., S616) in regulating its function . Similar phosphorylation events likely regulate plant DRP1A activity, particularly during developmental transitions or stress responses. Researchers investigating these modifications should consider combining immunoprecipitation using anti-DRP1A antibodies with phosphoproteomic analysis to identify regulatory sites.

Why might DRP1A antibody show inconsistent detection across different plant tissues?

Inconsistent DRP1A detection can result from several technical and biological factors:

Research has revealed that DRP1A functions in various developmental processes, including embryogenesis, cell plate formation, and trichome branching . This suggests tissue-specific regulation that may affect detection. When comparing DRP1A levels across tissues, researchers should normalize to appropriate loading controls and consider using multiple antibodies targeting different epitopes to ensure comprehensive detection.

How can researchers optimize DRP1A immunoprecipitation protocols for interaction studies?

Optimizing DRP1A immunoprecipitation requires careful consideration of several parameters:

• Buffer composition:

  • For membrane-associated interactions, include 0.5-1% digitonin or 0.5% NP-40

  • For stronger interactions, RIPA buffer may be suitable

  • Always include protease and phosphatase inhibitors

• Antibody coupling:

  • Direct coupling to beads (using BS3 or DMP) prevents antibody contamination in eluates

  • For weaker interactions, gentle cross-linking approaches are preferable

• Incubation conditions:

  • Overnight incubation at 4°C with gentle rotation

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Validation approaches:

  • Confirm IP efficiency by immunoblotting 5-10% of input, unbound, and elution fractions

  • Include IgG control and, when available, immunoprecipitation from drp1a mutant tissue

Based on research showing DRP1A interactions with DRP2B in endocytic processes , immunoprecipitation protocols should be designed to preserve membrane-associated protein complexes. The subcellular co-localization and intermolecular interaction between these proteins suggest that membrane solubilization conditions are critical for successful co-immunoprecipitation experiments.

What approaches can resolve contradictory findings between antibody-based detection and transcriptional analysis of DRP1A?

Researchers sometimes encounter discrepancies between protein levels detected by antibodies and mRNA expression. To resolve such contradictions:

• Validate antibody specificity:

  • Compare detection in wild-type vs. drp1a mutants

  • Perform peptide competition assays

  • Test multiple antibodies targeting different epitopes

• Assess post-transcriptional regulation:

  • Measure protein half-life using cycloheximide chase experiments

  • Investigate miRNA-mediated regulation

  • Examine polysome association of DRP1A transcripts

• Consider technical limitations:

  • Different sensitivities between RT-qPCR and immunoblotting

  • Non-linear relationship between transcript and protein

  • Tissue-specific translation efficiency

Research has demonstrated that while drp1a mutants show highly reduced DRP1A mRNA levels, protein detection using antibodies provides the definitive proof of null mutation status . This highlights the complementary nature of transcript and protein analysis. In some experimental systems, researchers have observed no differences in steady-state FLS2 mRNA or protein levels in drp1a mutants compared to wild-type plants, even though functional differences exist in receptor dynamics , emphasizing the importance of analyzing both static levels and dynamic processes.

How can DRP1A antibodies be used to compare dynamin-related protein functions across plant species?

DRP1A antibodies enable comparative studies across diverse plant species:

• Cross-species immunoblotting:

  • Assess conservation and divergence in protein size and abundance

  • Compare expression patterns across developmental stages

  • Evaluate responses to environmental stressors

• Immunolocalization studies:

  • Compare subcellular localization patterns

  • Assess recruitment to endocytic sites

  • Evaluate co-localization with conserved endocytic markers

• Experimental considerations:

  • Select antibodies with broad cross-reactivity profiles

  • Adjust protein extraction protocols for species-specific tissue composition

  • Validate specificity in each species through appropriate controls

The available anti-DRP1A antibodies show reactivity with a wide range of plant species including Arabidopsis thaliana, Brassica species, Spinacia oleracea, Cucumis sativus, Vitis vinifera, Solanum species, Populus trichocarpa, Medicago truncatula, Glycine max, and various cereal crops including Zea mays, Oryza sativa, Panicum virgatum, Setaria viridis, Sorghum bicolor, Triticum aestivum, and Hordeum vulgare . This broad reactivity stems from conservation of the immunizing peptide sequence, with the synthetic peptide used for immunization being 93% (14/15 amino acids) homologous with the sequence in the related protein DL1B (AT3G61760) .

What are the differences between plant DRP1A and mammalian Drp1, and how can researchers account for these in experimental design?

While sharing functional similarities, plant DRP1A and mammalian Drp1 exhibit important differences:

Research on mammalian Drp1 has revealed its involvement in neurodegenerative diseases (Alzheimer's, Huntington's, Parkinson's), cardiovascular diseases, and various cancers . In contrast, plant DRP1A functions primarily in developmental processes and immunity. When designing experiments using antibodies that might cross-react between plant and animal systems, researchers should carefully validate specificity and be aware that regulatory mechanisms likely differ significantly between kingdoms.

How might emerging nanobody technologies complement traditional DRP1A antibodies in plant research?

Nanobodies offer exciting new approaches for studying DRP1A dynamics:

• Advantages over traditional antibodies:

  • Smaller size (approximately 15 kDa vs. 150 kDa for IgG)

  • Greater epitope accessibility in complex structures

  • Potential for live-cell imaging when fused to fluorescent proteins

  • Greater stability under various experimental conditions

• Potential applications:

  • Live-cell tracking of DRP1A dynamics during endocytosis

  • Super-resolution microscopy with minimal linkage error

  • Manipulation of DRP1A function through targeted nanobody binding

  • Proximity-dependent labeling with nanobody-enzyme fusions

Recent research has developed nanobodies as versatile tools for proteomics, advanced microscopy, and live cell imaging of mammalian Drp1 . Similar approaches could be applied to plant DRP1A, enabling unprecedented insights into its dynamic behavior during cell division, endocytosis, and immune responses. When combining nanobody and traditional antibody approaches, researchers should consider epitope competition and validate that nanobody binding doesn't interfere with DRP1A function.

What methodologies can integrate DRP1A antibodies with emerging single-cell technologies in plant research?

Integrating DRP1A antibody detection with single-cell approaches presents exciting opportunities:

• Single-cell protein profiling:

  • Combine flow cytometry with intracellular DRP1A staining

  • Apply mass cytometry (CyTOF) with metal-conjugated anti-DRP1A antibodies

  • Implement microfluidic platforms for single-cell Western blotting

• Spatial analysis:

  • Employ imaging mass cytometry to map DRP1A distribution in tissues

  • Use multiplexed immunofluorescence to correlate DRP1A with other proteins

  • Apply proximity ligation assays to detect specific interactions in situ

• Experimental considerations:

  • Optimize cell isolation protocols to maintain protein integrity

  • Validate antibody specificity in single-cell formats

  • Develop appropriate normalization standards for quantitative analysis

While single-cell approaches are still emerging in plant science, DRP1A's involvement in fundamental cellular processes makes it an ideal target for techniques that can resolve cell-to-cell variability. The observation that DRP1A functions in diverse processes including embryogenesis, cell plate formation, trichome branching, and immunity suggests that its activity may vary substantially between cell types, making single-cell analysis particularly informative.