PSKR2 Antibody

Basic Research Questions

What experimental designs are optimal for detecting PSKR2 in plant signaling studies?

• Methodology: Combine co-immunoprecipitation (co-IP) with calcium-dependent kinase activity assays, as PSKR2 interacts with kinases like CPK28 in ligand-dependent signaling cascades . For tissue-specific localization, use confocal microscopy with fluorescently tagged PSKR2 antibodies in transgenic lines (e.g., Arabidopsis or tomato).

• Key controls: Include PSKR2 knockout mutants to confirm antibody specificity, and validate interactions using Förster resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) .

How can researchers validate PSKR2 antibody specificity in cross-species studies?

• Approach:

  • Perform Western blotting against protein extracts from phylogenetically diverse species (e.g., Arabidopsis, tomato, rice).

  • Use epitope mapping to identify conserved binding regions (e.g., extracellular leucine-rich repeats or kinase domains) .

  • Validate via genetic complementation: Express PSKR2 orthologs in heterologous systems (e.g., Nicotiana benthamiana) and test antibody reactivity .

What are common pitfalls in PSKR2 antibody-based assays, and how to mitigate them?

• Challenges:

  • Non-specific binding due to homology with PSKR1 .

  • Ligand-induced conformational changes altering epitope accessibility .

• Solutions:

  • Pre-adsorb antibodies with PSKR1 recombinant protein.

  • Optimize fixation protocols to preserve native conformations during immunolocalization .

Advanced Research Questions

How to resolve data contradictions in PSKR2-mediated growth-defense trade-offs?

• Case example: Conflicting reports on PSKR2’s role in immunity (pro-defense vs. susceptibility) .

• Methodological strategy:

  • Contextualize experimental conditions (e.g., pathogen strain, nitrogen availability).

  • Quantify phosphorylation states of downstream targets (e.g., GS2 at S334/S360) using Phos-tag gels or mass spectrometry .

  • Conduct transcriptome profiling to correlate PSKR2 activity with defense/growth marker genes (e.g., PR1 vs. ribosomal biogenesis genes) .

Does PSKR2 exhibit functional redundancy with PSKR1, and how to dissect this?

• Experimental framework:

  • Generate PSKR1/PSKR2 double mutants and compare phenotypes (e.g., root growth, pathogen susceptibility) to single mutants .

  • Use structural modeling to identify divergent domains (e.g., activation loops) and design domain-swap transgenes for functional testing .

  • Key finding: In Arabidopsis, PSKR1 dominates growth regulation, while PSKR2 partially compensates in immune responses .

How to investigate PSKR2’s role in phosphorylation-dependent signaling networks?

• Workflow:

  • Identify phosphorylation sites via mutagenesis (e.g., Ser→Ala/Asp substitutions) and test kinase activity in vitro .

  • Map interactors using TurboID proximity labeling in planta, followed by LC-MS/MS .

  • Data integration: Build a phosphorylation-regulated interactome table:

What advanced techniques improve PSKR2 antibody specificity for structural studies?

• Innovative methods:

  • Phage display-guided engineering: Design single-chain variable fragments (scFvs) with >10-fold specificity over PSKR1 by counter-selecting against PSKR1 epitopes .

  • Cryo-EM with nanodisc-embedded PSKR2: Resolve extracellular domain conformations in lipid bilayers to guide epitope selection .

Methodological Best Practices

• For flow cytometry: Use TrueStain Monocyte Blocker to reduce non-specific binding in myeloid-rich tissues .

• In kinase assays: Include Ca²⁺/Mg²⁺ chelators (e.g., EDTA) only if studying calcium-independent activity .